Plant regulatory elements derived from medicago truncatula 3&#39;utr sequences, and uses thereof

ABSTRACT

The invention provides recombinant DNA molecules and constructs, as well as their nucleotide sequences, useful for modulating gene expression in plants. The invention also provides transgenic plants, plant cells, plant parts, and seeds comprising a recombinant DNA molecule comprising a DNA molecule operably linked to heterologous transcribable DNA molecule, as are methods of their use.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 61/785,268, filed Mar. 14, 2013, which is incorporated by referenceherein in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“MONS332US.txt”, which is 52.7 kilobytes (as measured in MicrosoftWindows®) and was created on Mar. 11, 2014, is filed herewith byelectronic submission and is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to the field of plant molecular biology, plantgenetic engineering, and DNA molecules useful for modulating geneexpression in plants.

BACKGROUND

Regulatory elements are genetic elements that regulate gene activity bymodulating the transcription of an operably linked transcribable DNAmolecule. Such elements may include promoters, leaders, introns, and 3′untranslated regions and are useful in the field of plant molecularbiology and plant genetic engineering.

SUMMARY OF THE INVENTION

The invention provides novel regulatory elements for use in plants, andconstructs comprising the regulatory elements. The invention alsoprovides transgenic plant cells, plants, and seeds comprising theregulatory elements. In one embodiment disclosed herein, the regulatoryelements are operably linked to a transcribable DNA molecule. In certainembodiments, the transcribable DNA molecule is heterologous with respectto the regulatory sequence. Also provided herein are methods for makingand using the regulatory elements disclosed herein, including constructscomprising the regulatory elements, and the transgenic plant cells,plants, and seeds comprising the regulatory elements operably linked toa transcribable DNA molecule that is heterologous with respect to theregulatory element.

Thus, in one aspect, the invention provides a recombinant DNA moleculecomprising a DNA sequence selected from the group consisting of: (a) aDNA sequence with at least about 85 percent sequence identity to any ofSEQ ID NOs: 1-37; (b) a DNA sequence comprising any of SEQ ID NOs: 1-37;and (c) a fragment of any of SEQ ID NOs: 1-37, wherein the fragment hasgene-regulatory activity; wherein the DNA sequence is operably linked toa heterologous transcribable DNA molecule. By “heterologoustranscribable DNA molecule,” it is meant that the transcribable DNAmolecule is heterologous with respect to the DNA sequence to which it isoperably linked. In specific embodiments, the recombinant DNA moleculecomprises a DNA sequence having at least 90 percent, at least 91percent, at least 92 percent, at least 93 percent, at least 94 percent,at least 95 percent, at least 96 percent, at least 97 percent, at least98 percent, or at least 99 percent sequence identity to the DNA sequenceof any of SEQ ID NOs: 1-37. In particular embodiments, the heterologoustranscribable DNA molecule comprises a gene of agronomic interest, suchas a gene capable of providing herbicide resistance or pest resistancein plants. In still other embodiments, the invention provides aconstruct comprising a recombinant DNA molecule as provided herein.

In another aspect, provided herein are transgenic plant cells comprisinga recombinant DNA molecule comprising a DNA sequence selected from thegroup consisting of: (a) a DNA sequence with at least about 85 percentsequence identity to any of SEQ ID NOs: 1-37; (b) a DNA sequencecomprising any of SEQ ID NOs: 1-37; and (c) a fragment of any of SEQ IDNOs: 1-37, wherein the fragment has gene-regulatory activity; whereinthe DNA sequence is operably linked to a heterologous transcribable DNAmolecule. In certain embodiments, the transgenic plant cell is amonocotyledonous plant cell. In other embodiments, the transgenic plantcell is a dicotyledonous plant cell.

In still yet another aspect, further provided herein is a transgenicplant, or part thereof, comprising a recombinant DNA molecule comprisinga DNA sequence selected from the group consisting of: a) a DNA sequencewith at least 85 percent sequence identity to any of SEQ ID NOs: 1-37;b) a DNA sequence comprising any of SEQ ID NOs: 1-37; and c) a fragmentof any of SEQ ID NOs: 1-37, wherein the fragment has gene-regulatoryactivity; wherein the DNA sequence is operably linked to a heterologoustranscribable DNA molecule. In specific embodiments, the transgenicplant is a progeny plant of any generation relative to a startingtransgenic plant and comprises the recombinant DNA molecule. Atransgenic seed comprising the recombinant DNA molecule that producessuch a transgenic plant when grown is also provided herein.

In another aspect, the invention provides a method of producing acommodity product comprising obtaining a transgenic plant or partthereof containing a recombinant DNA molecule of the invention andproducing the commodity product therefrom. In one embodiment, thecommodity product is processed seeds, grains, plant parts, and meal.

In still yet another aspect, the invention provides a method ofproducing a transgenic plant comprising a recombinant DNA molecule ofthe invention comprising transforming a plant cell with the recombinantDNA molecule of the invention to produce a transformed plant cell andregenerating a transgenic plant from the transformed plant cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows expression cassette configurations of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NOs: 1-30, 38-41, 49 and 56 are 3′ UTR sequences.

SEQ ID NOs: 31, 35, 42, 47, 48, 50, 51, 52, 53, 54 and 55 are DNAsequences of regulatory expression element groups (EXPs) comprising apromoter sequence operably linked 5′ to a leader sequence, which isoperably linked 5′ to an intron sequence; or a promoter sequenceoperably linked 5′ to a leader sequence.

SEQ ID NOs: 32, 36, and 43 are promoter sequences.

SEQ ID NOs: 33 and 37 are leader sequences.

SEQ ID NO: 34 is an intron sequence.

SEQ ID NO: 44 is a coding sequence for β-glucuronidase (GUS) thatpossesses a processable intron.

SEQ ID NOs: 45 and 46 are luciferase coding sequences.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides DNA molecules having gene-regulatory activity inplants. The nucleotide sequences of these DNA molecules are provided asSEQ ID NOs: 1-37. These DNA molecules are capable of affecting theexpression of an operably linked transcribable DNA molecule in planttissues, and therefore regulating gene expression of an operably linkedtransgene in transgenic plants. The invention also provides methods ofmodifying, producing, and using the same. The invention also providescompositions that include transgenic plant cells, plants, plant parts,and seeds containing the recombinant DNA molecules of the invention, andmethods for preparing and using the same.

The following definitions and methods are provided to better define theinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

DNA Molecules

As used herein, the term “DNA” or “DNA molecule” refers to adouble-stranded DNA molecule of genomic or synthetic origin, i.e., apolymer of deoxyribonucleotide bases. As used herein, the term “DNAsequence” to the nucleotide sequence of a DNA molecule. The nomenclatureused herein corresponds to that of Title 37 of the United States Code ofFederal Regulations § 1.822, and set forth in the tables in WIPOStandard ST.25 (1998), Appendix 2, Tables 1 and 3.

As used herein, a “recombinant DNA molecule” is a DNA moleculecomprising a combination of DNA molecules that would not naturally occurtogether without human intervention. For instance, a recombinant DNAmolecule may be a DNA molecule that is comprised of at least two DNAmolecules heterologous with respect to each other, a DNA molecule thatcomprises a DNA sequence that deviates from DNA sequences that exist innature, or a DNA molecule that has been incorporated into a host cell'sDNA by genetic transformation.

As used herein, the term “sequence identity” refers to the extent towhich two optimally aligned DNA sequences are identical. An optimalsequence alignment is created by manually aligning two sequences, e.g.,a reference sequence and another DNA sequence, to maximize the number ofnucleotide matches in the sequence alignment with appropriate internalnucleotide insertions, deletions, or gaps. As used herein, the term“reference sequence” refers to a DNA sequence provided as SEQ ID NOs:1-37.

As used herein, the term “percent sequence identity” or “percentidentity” or “% identity” is the identity fraction multiplied by 100.The “identity fraction” for a DNA sequence optimally aligned with areference sequence is the number of nucleotide matches in the optimalalignment, divided by the total number of nucleotides in the referencesequence, e.g., the total number of nucleotides in the full length ofthe entire reference sequence. Thus, one embodiment of the inventionprovides a DNA molecule comprising a DNA sequence that, when optimallyaligned to a reference sequence, provided herein as SEQ ID NOs: 1-37,has at least about 85 percent identity, at least about 86 percentidentity, at least about 87 percent identity, at least about 88 percentidentity, at least about 89 percent identity, at least about 90 percentidentity, at least about 91 percent identity, at least about 92 percentidentity, at least about 93 percent identity, at least about 94 percentidentity, at least about 95 percent identity, at least about 96 percentidentity, at least about 97 percent identity, at least about 98 percentidentity, at least about 99 percent identity, or at least about 100percent identity to the reference sequence.

Regulatory Elements

Regulatory elements such as promoters, leaders, enhancers, introns, andtranscription termination regions (or 3′ UTRs) play an integral part inthe overall expression of genes in living cells. The term “regulatoryelement,” as used herein, refers to a DNA molecule havinggene-regulatory activity. The term “gene-regulatory activity,” as usedherein, refers to the ability to affect the expression of an operablylinked transcribable DNA molecule, for instance by affecting thetranscription and/or translation of the operably linked transcribableDNA molecule. Regulatory elements, such as promoters, leaders,enhancers, introns and 3′ UTRs that function in plants are thereforeuseful for modifying plant phenotypes through genetic engineering.

As used herein, a “regulatory expression element group” or “EXP”sequence may refer to a group of operably linked regulatory elements,such as enhancers, promoters, leaders, and introns. Thus, a regulatoryexpression element group may be comprised, for instance, of a promoteroperably linked 5′ to a leader sequence, which is in turn operablylinked 5′ to an intron sequence.

Regulatory elements may be characterized by their gene expressionpattern, e.g., positive and/or negative effects such as constitutive,temporal, spatial, developmental, tissue, environmental, physiological,pathological, cell cycle, and/or chemically responsive expression, andany combination thereof, as well as by quantitative or qualitativeindications. As used herein, a “gene expression pattern” is any patternof transcription of an operably linked DNA molecule into a transcribedRNA molecule. The transcribed RNA molecule may be translated to producea protein molecule or may provide an antisense or other regulatory RNAmolecule, such as double-stranded RNA (dsRNA), a transfer RNA (tRNA), aribosomal RNA (rRNA), a microRNA (miRNA), and the like.

As used herein, the term “protein expression” is any pattern oftranslation of a transcribed RNA molecule into a protein molecule.Protein expression may be characterized by its temporal, spatial,developmental, or morphological qualities, as well as by quantitative orqualitative indications.

A promoter is useful as a regulatory element for modulating theexpression of an operably linked transcribable DNA molecule. As usedherein, the term “promoter” refers generally to a DNA molecule that isinvolved in recognition and binding of RNA polymerase II and otherproteins, such as trans-acting transcription factors, to initiatetranscription. A promoter may be initially identified from the 5′untranslated region (5′ UTR) of a gene. Alternately, promoters may besynthetically produced or manipulated DNA molecules. Promoters may alsobe chimeric. Chimeric promoters are produced through the fusion of twoor more heterologous DNA molecules. Promoters useful in practicing theinvention include SEQ ID NOs: 32 and 36, including fragments or variantsthereof. In specific embodiments of the invention, the claimed DNAmolecules and any variants or derivatives thereof as described herein,are further defined as comprising promoter activity, i.e., are capableof acting as a promoter in a host cell, such as in a transgenic plant.In still further specific embodiments, a fragment may be defined asexhibiting promoter activity possessed by the starting promoter moleculefrom which it is derived, or a fragment may comprise a “minimalpromoter” which provides a basal level of transcription and is comprisedof a TATA box or equivalent DNA sequence for recognition and binding ofthe RNA polymerase II complex for initiation of transcription.

In one embodiment, fragments of a promoter sequence disclosed herein areprovided. Promoter fragments may comprise promoter activity, asdescribed above, and may be useful alone or in combination with otherpromoters and promoter fragments, such as in constructing chimericpromoters, or in combination with other EXPs and EXP fragments. Inspecific embodiments, fragments of a promoter are provided comprising atleast about 50, at least about 75, at least about 95, at least about100, at least about 125, at least about 150, at least about 175, atleast about 200, at least about 225, at least about 250, at least about275, at least about 300, at least about 500, at least about 600, atleast about 700, at least about 750, at least about 800, at least about900, or at least about 1000 contiguous nucleotides, or longer, of a DNAmolecule having promoter activity as disclosed herein. Methods forproducing such fragments from a starting promoter molecule are wellknown in the art.

Compositions derived from any of the promoters presented as SEQ ID NOs:32 and 36, such as internal or 5′ deletions, for example, can beproduced using methods well known in the art to improve or alterexpression, including by removing elements that have either positive ornegative effects on expression; duplicating elements that have positiveor negative effects on expression; and/or duplicating or removingelements that have tissue- or cell-specific effects on expression.Compositions derived from any of the promoters presented as SEQ ID NOs:32 and 36 comprised of 3′ deletions in which the TATA box element orequivalent sequence thereof and downstream sequence is removed can beused, for example, to make enhancer elements. Further deletions can bemade to remove any elements that have positive or negative; tissuespecific; cell specific; or timing specific (such as, but not limitedto, circadian rhythm) effects on expression. Any of the promoterspresented as SEQ ID NOs: 32 and 36 and fragments or enhancers derivedtherefrom can be used to make chimeric transcriptional regulatoryelement compositions.

In accordance with the invention, a promoter or promoter fragment may beanalyzed for the presence of known promoter elements, i.e., DNA sequencecharacteristics, such as a TATA box and other known transcription factorbinding site motifs. Identification of such known promoter elements maybe used by one of skill in the art to design variants of the promoterhaving a similar expression pattern to the original promoter.

As used herein, the term “leader” refers to a DNA molecule identifiedfrom the untranslated 5′ region (5′ UTR) of a gene and defined generallyas a nucleotide segment between the transcription start site (TSS) andthe protein coding sequence start site. Alternately, leaders may besynthetically produced or manipulated DNA elements. A leader can be usedas a 5′ regulatory element for modulating expression of an operablylinked transcribable DNA molecule. Leader molecules may be used with aheterologous promoter or with their native promoter. Leaders useful inpracticing the invention include SEQ ID NOs: 33 and 37 or fragments orvariants thereof. In specific embodiments, such DNA sequences may bedefined as being capable of acting as a leader in a host cell,including, for example, a transgenic plant cell. In one embodiment, suchDNA sequences may be decoded as comprising leader activity.

The leader sequences presented as SEQ ID NOs: 33 and 37 may be comprisedof regulatory elements, or may adopt secondary structures that can havean effect on transcription or translation of an operably linkedtranscribable DNA molecule. The leader sequences presented as SEQ IDNOs: 33 and 37 can be used in accordance with the invention to makechimeric regulatory elements that affect transcription or translation ofan operably linked DNA molecule.

As used herein, the term “intron” refers to a DNA molecule that may beidentified from a gene and may be defined generally as a region splicedout during messenger RNA (mRNA) processing prior to translation.Alternately, an intron may be a synthetically produced or manipulatedDNA element. An intron may contain enhancer elements that effect thetranscription of operably linked genes. An intron may be used as aregulatory element for modulating expression of an operably linkedtranscribable DNA molecule. A construct may comprise an intron, and theintron may or may not be heterologous with respect to the transcribableDNA molecule. Examples of introns in the art include the rice actinintron and the corn HSP70 intron.

In plants, the inclusion of some introns in gene constructs leads toincreased mRNA and protein accumulation relative to constructs lackingthe intron. This effect has been termed “intron mediated enhancement”(IME) of gene expression. Introns known to stimulate expression inplants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, andUbi1), in rice genes (e.g., tpi) and in dicotyledonous plant genes likethose from petunia (e.g., rbcS), potato (e.g., st-ls1) and fromArabidopsis thaliana (e.g., ubq3 and pat1). It has been shown thatdeletions or mutations within the splice sites of an intron reduce geneexpression, indicating that splicing might be needed for IME. However,IME in dicotyledonous plants has been shown by point mutations withinthe splice sites of the pat1 gene from A. thaliana. Multiple uses of thesame intron in one plant has been shown, in certain circumstances, toexhibit disadvantages. In those cases, it is necessary to have acollection of basic control elements for the construction of appropriaterecombinant DNA elements.

Introns useful in practicing the invention include SEQ IN NO: 34.Compositions derived from the intron presented as SEQ ID NO: 34 can becomprised of internal deletions or duplications of cis-regulatoryelements; and/or alterations of the 5′ and 3′ DNA sequences comprisingthe intron/exon splice junctions can be used to improve expression orspecificity of expression when operably linked to a promoter+leader orchimeric promoter+leader and coding sequence. When modifying intron/exonboundary sequences, it may be beneficial to avoid using the nucleotidesequence AT or the nucleotide A just prior to the 5′ end of the splicesite (GT) and the nucleotide G or the nucleotide sequence TG,respectively, just after the 3′ end of the splice site (AG) to eliminatethe potential of unwanted start codons from being formed duringprocessing of the messenger RNA into the final transcript. The DNAsequence around the 5′ or 3′ end splice junction sites of the intron canthus be modified in this manner. Intron and intron variants altered asdescribed herein and through methods known in the art can be testedempirically as described in the working examples to determine theintron's effect on expression of an operably linked DNA molecule.Alterations of the 5′ and 3′ regions comprising the intron/exon splicejunction can also be made to reduce the potential for introduction offalse start and stop codons being produced in the resulting transcriptafter processing and splicing of the messenger RNA. The introns can betested empirically as described in the working examples to determine theintron's effect on expression of a transgene.

As used herein, the terms “3′ transcription termination molecule,” “3′untranslated region” or “3′ UTR” refer to a DNA molecule that is usedduring transcription to the untranslated region of the 3′ portion of anmRNA molecule. The 3′ untranslated region of an mRNA molecule may begenerated by specific cleavage and 3′ polyadenylation, also known as apolyA tail. A 3′ UTR may be operably linked to and located downstream ofa transcribable DNA molecule and may include a polyadenylation signaland other regulatory signals capable of affecting transcription, mRNAprocessing, or gene expression. PolyA tails are thought to function inmRNA stability and in initiation of translation. Examples of 3′transcription termination molecules in the art are the nopaline synthase3′ region; wheat hsp17 3′ region, pea rubisco small subunit 3′ region,cotton E6 3′ region, and the coixin 3′ UTR.

3′ UTRs typically find beneficial use for the recombinant expression ofspecific DNA molecules. A weak 3′ UTR has the potential to generateread-through, which may affect the expression of the DNA moleculelocated in the neighboring expression cassettes. Appropriate control oftranscription termination can prevent read-through into DNA sequences(e.g., other expression cassettes) localized downstream and can furtherallow efficient recycling of RNA polymerase to improve gene expression.Efficient termination of transcription (release of RNA polymerase IIfrom the DNA) is prerequisite for re-initiation of transcription andthereby directly affects the overall transcript level. Subsequent totranscription termination, the mature mRNA is released from the site ofsynthesis and template transported to the cytoplasm. Eukaryotic mRNAsare accumulated as poly(A) forms in vivo, making it difficult to detecttranscriptional termination sites by conventional methods. However,prediction of functional and efficient 3′ UTRs by bioinformatics methodscan be difficult in that there are few conserved DNA sequences thatwould allow for easy prediction of an effective 3′ UTR.

From a practical standpoint, it is typically beneficial that a 3′ UTRused in an expression cassette possesses the following characteristics.The 3′ UTR should be able to efficiently and effectively terminatetranscription of the transgene and prevent read-through of thetranscript into any neighboring DNA sequence, which can be comprised ofanother expression cassette as in the case of multiple expressioncassettes residing in one transfer DNA (T-DNA), or the neighboringchromosomal DNA into which the T-DNA has inserted. In plantbiotechnology, the 3′ UTR is often used for priming of amplificationreactions of reverse transcribed RNA extracted from the transformedplant and used to: (1) assess the transcriptional activity or expressionof the expression cassette once integrated into the plant chromosome;(2) assess the copy number of insertions within the plant DNA; and (3)assess zygosity of the resulting seed after breeding. The 3′ UTR is alsoused in amplification reactions of DNA extracted from the transformedplant to characterize the intactness of the inserted cassette.

As used herein, the term “enhancer” or “enhancer element” refers to acis-acting regulatory element, a.k.a. cis-element, which confers anaspect of the overall expression pattern, but is usually insufficientalone to drive transcription, of an operably linked transcribable DNAmolecule. Unlike promoters, enhancer elements do not usually include atranscription start site (TSS) or TATA box or equivalent DNA sequence. Apromoter or promoter fragment may naturally comprise one or moreenhancer elements that affect the transcription of an operably linkedtranscribable DNA molecule. An enhancer element may also be fused to apromoter to produce a chimeric promoter cis-element, which confers anaspect of the overall modulation of gene expression.

Many promoter enhancer elements are believed to bind DNA-bindingproteins and/or affect DNA topology, producing local conformations thatselectively allow or restrict access of RNA polymerase to the DNAtemplate or that facilitate selective opening of the double helix at thesite of transcriptional initiation. An enhancer element may function tobind transcription factors that regulate transcription. Some enhancerelements bind more than one transcription factor, and transcriptionfactors may interact with different affinities with more than oneenhancer domain. Enhancer elements can be identified by a number oftechniques, including deletion analysis, i.e., deleting one or morenucleotides from the 5′ end or internal to a promoter; DNA bindingprotein analysis using DNase I footprinting, methylation interference,electrophoresis mobility-shift assays, in vivo genomic footprinting byligation-mediated polymerase chain reaction (PCR), and otherconventional assays; or by DNA sequence similarity analysis using knowncis-element motifs or enhancer elements as a target sequence or targetmotif with conventional DNA sequence comparison methods, such as BLAST.The fine structure of an enhancer domain can be further studied bymutagenesis (or substitution) of one or more nucleotides or by otherconventional methods known in the art. Enhancer elements can be obtainedby chemical synthesis or by isolation from regulatory elements thatinclude such elements, and they can be synthesized with additionalflanking nucleotides that contain useful restriction enzyme sites tofacilitate subsequence manipulation. Thus, the design, construction, anduse of enhancer elements according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the invention.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither the first nor the second DNA molecule would normally be found inthat configuration, i.e., fused to the other. The chimeric DNA moleculeis thus a new DNA molecule not otherwise normally found in nature. Asused herein, the term “chimeric promoter” refers to a promoter producedthrough such manipulation of DNA molecules. A chimeric promoter maycombine two or more DNA fragments, for example, the fusion of a promoterto an enhancer element. Thus, the design, construction, and use ofchimeric promoters according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the present invention.

As used herein, the term “variant” refers to a second DNA molecule, suchas a regulatory element, that is similar in composition, but notidentical to, a first DNA molecule, and wherein the second DNA moleculestill maintains the general functionality, i.e., same or similarexpression pattern, for instance through more or less or equivalenttranscriptional or translational activity, of the first DNA molecule. Avariant may be a shorter or truncated version of the first DNA moleculeand/or an altered version of the sequence of the first DNA molecule,such as one with different restriction enzyme sites and/or internaldeletions, substitutions, and/or insertions. A “variant” can alsoencompass a regulatory element having a nucleotide sequence comprising asubstitution, deletion, and/or insertion of one or more nucleotides of areference sequence, wherein the derivative regulatory element has moreor less or equivalent transcriptional or translational activity than thecorresponding parent regulatory molecule. Regulatory element “variants”also encompass variants arising from mutations that occur during or as aresult of bacterial and plant cell transformation. In the invention, aDNA sequence provided as SEQ ID NOs: 1-37 may be used to create variantsthat are in similar in composition, but not identical to, the DNAsequence of the original regulatory element, while still maintaining thegeneral functionality, i.e., the same or similar expression pattern, ofthe original regulatory element. Production of such variants of theinvention is well within the ordinary skill of the art in light of thedisclosure and is encompassed within the scope of the invention.

Chimeric regulatory elements can be designed to comprise variousconstituent elements which may be operatively linked by various methodsknown in the art, such as restriction enzyme digestion and ligation,ligation independent cloning, modular assembly of PCR products duringamplification, or direct chemical synthesis of the regulatory element,as well as other methods known in the art. The resulting variouschimeric regulatory elements can be comprised of the same, or variantsof the same, constituent elements but differ in the DNA sequence or DNAsequences that comprise the linking DNA sequence or sequences that allowthe constituent parts to be operatively linked. In the invention, a DNAsequence provided as SEQ ID NOs: 1-30 or 31-37 may provide a regulatoryelement reference sequence, wherein the constituent elements thatcomprise the reference sequence may be joined by methods known in theart and may comprise substitutions, deletions, and/or insertions of oneor more nucleotides or mutations that naturally occur in bacterial andplant cell transformation.

The efficacy of the modifications, duplications, or deletions describedherein on the desired expression aspects of a particular transcribableDNA molecule may be tested empirically in stable and transient plantassays, such as those described in the working examples herein, so as tovalidate the results, which may vary depending upon the changes made andthe goal of the change in the starting DNA molecule.

Constructs

As used herein, the term “construct” means any recombinant DNA moleculesuch as a plasmid, cosmid, virus, phage, or linear or circular DNA orRNA molecule, derived from any source, capable of genomic integration orautonomous replication, comprising a DNA molecule where at least one DNAmolecule has been linked to another DNA molecule in a functionallyoperative manner, i.e., operably linked. As used herein, the term“vector” means any construct that may be used for the purpose oftransformation, i.e., the introduction of heterologous DNA or RNA into ahost cell. A construct typically includes one or more expressioncassettes. As used herein, an “expression cassette” refers to a DNAmolecule comprising at least a transcribable DNA molecule operablylinked to one or more regulatory elements, typically at least a promoterand a 3′ UTR.

As used herein, the term “operably linked” refers to a first DNAmolecule joined to a second DNA molecule, wherein the first and secondDNA molecules are arranged so that the first DNA molecule affects thefunction of the second DNA molecule. The two DNA molecules may or maynot be part of a single contiguous DNA molecule and may or may not beadjacent. For example, a promoter is operably linked to a transcribableDNA molecule if the promoter is capable of affecting the transcriptionor translation of the transcribable DNA molecule.

The constructs of the invention may be provided, in one embodiment, asdouble tumor-inducing (Ti) plasmid border constructs that have the rightborder (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of theTi plasmid isolated from Agrobacterium tumefaciens comprising a T-DNAthat, along with transfer molecules provided by the A. tumefacienscells, permit the integration of the T-DNA into the genome of a plantcell (see, e.g., U.S. Pat. No. 6,603,061). The constructs may alsocontain the plasmid backbone DNA segments that provide replicationfunction and antibiotic selection in bacterial cells, e.g., anEscherichia coli origin of replication such as ori322, a broad hostrange origin of replication such as oriV or oriRi, and a coding regionfor a selectable marker such as Spec/Strp that encodes for Tn7aminoglycoside adenyltransferase (aadA) conferring resistance tospectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectablemarker gene. For plant transformation, the host bacterial strain isoften A. tumefaciens ABI, C58, or LBA4404; however, other strains knownto those skilled in the art of plant transformation can function in theinvention.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the transcribable DNA molecule istranscribed into a functional mRNA molecule that is translated andexpressed as a protein. For the practice of the invention, conventionalcompositions and methods for preparing and using constructs and hostcells are well known to one skilled in the art. For example, typicalvectors useful for expression of nucleic acids in higher plants are wellknown in the art and include vectors derived from the Ti plasmid ofAgrobacterium tumefaciens and the pCaMVCN transfer control vector.

Various regulatory elements may be included in a construct, includingany of those provided herein. Any such regulatory elements may beprovided in combination with other regulatory elements. Suchcombinations can be designed or modified to produce desirable regulatoryfeatures. In one embodiment, constructs of the invention comprise atleast one regulatory element operably linked to a transcribable DNAmolecule operably linked to a 3′ UTR.

Constructs of the invention may include any promoter or leader providedherein or known in the art. For example, a promoter of the invention maybe operably linked to a heterologous non-translated 5′ leader such asone derived from a heat shock protein gene. Alternatively, a leader ofthe invention may be operably linked to a heterologous promoter such asthe Cauliflower Mosaic Virus 35S transcript promoter.

Expression cassettes may also include a transit peptide coding sequencethat encodes a peptide that is useful for sub-cellular targeting of anoperably linked protein, particularly to a chloroplast, leucoplast, orother plastid organelle; mitochondria; peroxisome; vacuole; or anextracellular location. Many chloroplast-localized proteins areexpressed from nuclear genes as precursors and are targeted to thechloroplast by a chloroplast transit peptide (CTP). Examples of suchisolated chloroplast proteins include, but are not limited to, thoseassociated with the small subunit (SSU) of ribulose-1,5,-bisphosphatecarboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvestingcomplex protein I and protein II, thioredoxin F, and enolpyruvylshikimate phosphate synthase (EPSPS). Chloroplast transit peptides aredescribed, for example, in U.S. Pat. No. 7,193,133. It has beendemonstrated that non-chloroplast proteins may be targeted to thechloroplast by the expression of a heterologous CTP operably linked tothe transcribable DNA molecule encoding non-chloroplast proteins.

Transcribable DNA Molecules

As used herein, the term “transcribable DNA molecule” refers to any DNAmolecule capable of being transcribed into a RNA molecule, including,but not limited to, those having protein coding sequences and thoseproducing RNA molecules having sequences useful for gene suppression.The type of DNA molecule can include, but is not limited to, a DNAmolecule from the same plant, a DNA molecule from another plant, a DNAmolecule from a different organism, or a synthetic DNA molecule, such asa DNA molecule containing an antisense message of a gene, or a DNAmolecule encoding an artificial, synthetic, or otherwise modifiedversion of a transgene. Exemplary transcribable DNA molecules forincorporation into constructs of the invention include, e.g., DNAmolecules or genes from a species other than the species into which theDNA molecule is incorporated or genes that originate from, or arepresent in, the same species, but are incorporated into recipient cellsby genetic engineering methods rather than classical breedingtechniques.

A “transgene” refers to a transcribable DNA molecule heterologous to ahost cell at least with respect to its location in the host cell genomeand/or a transcribable DNA molecule artificially incorporated into ahost cell's genome in the current or any prior generation of the cell.

A regulatory element, such as a promoter of the invention, may beoperably linked to a transcribable DNA molecule that is heterologouswith respect to the regulatory element. As used herein, the term“heterologous” refers to the combination of two or more DNA moleculeswhen such a combination is not normally found in nature. For example,the two DNA molecules may be derived from different species and/or thetwo DNA molecules may be derived from different genes, e.g., differentgenes from the same species or the same genes from different species. Aregulatory element is thus heterologous with respect to an operablylinked transcribable DNA molecule if such a combination is not normallyfound in nature, i.e., the transcribable DNA molecule does not naturallyoccur operably linked to the regulatory element.

The transcribable DNA molecule may generally be any DNA molecule forwhich expression of a transcript is desired. Such expression of atranscript may result in translation of the resulting mRNA molecule, andthus protein expression. Alternatively, for example, a transcribable DNAmolecule may be designed to ultimately cause decreased expression of aspecific gene or protein. In one embodiment, this may be accomplished byusing a transcribable DNA molecule that is oriented in the antisensedirection. One of ordinary skill in the art is familiar with using suchantisense technology. Any gene may be negatively regulated in thismanner, and, in one embodiment, a transcribable DNA molecule may bedesigned for suppression of a specific gene through expression of adsRNA, siRNA or miRNA molecule.

Thus, one embodiment of the invention is a recombinant DNA moleculecomprising a regulatory element of the invention, such as those providedas SEQ ID NOs: 1-37, operably linked to a heterologous transcribable DNAmolecule so as to modulate transcription of the transcribable DNAmolecule at a desired level or in a desired pattern when the constructis integrated in the genome of a transgenic plant cell. In oneembodiment, the transcribable DNA molecule comprises a protein-codingregion of a gene and in another embodiment the transcribable DNAmolecule comprises an antisense region of a gene.

Genes of Agronomic Interest

A transcribable DNA molecule may be a gene of agronomic interest. Asused herein, the term “gene of agronomic interest” refers to atranscribable DNA molecule that, when expressed in a particular planttissue, cell, or cell type, confers a desirable characteristic. Theproduct of a gene of agronomic interest may act within the plant inorder to cause an effect upon the plant morphology, physiology, growth,development, yield, grain composition, nutritional profile, disease orpest resistance, and/or environmental or chemical tolerance or may actas a pesticidal agent in the diet of a pest that feeds on the plant. Inone embodiment of the invention, a regulatory element of the inventionis incorporated into a construct such that the regulatory element isoperably linked to a transcribable DNA molecule that is a gene ofagronomic interest. In a transgenic plant containing such a construct,the expression of the gene of agronomic interest can confer a beneficialagronomic trait. A beneficial agronomic trait may include, for example,but is not limited to, herbicide tolerance, insect control, modifiedyield, disease resistance, pathogen resistance, modified plant growthand development, modified starch content, modified oil content, modifiedfatty acid content, modified protein content, modified fruit ripening,enhanced animal and human nutrition, biopolymer productions,environmental stress resistance, pharmaceutical peptides, improvedprocessing qualities, improved flavor, hybrid seed production utility,improved fiber production, and desirable biofuel production.

Examples of genes of agronomic interest known in the art include thosefor herbicide resistance (U.S. Pat. Nos. 6,803,501; 6,448,476;6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and5,463,175), increased yield (U.S. Pat. Nos. RE38,446; 6,716,474;6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211;6,235,971; 6,222,098; and 5,716,837), insect control (U.S. Pat. Nos.6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030;6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756;6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949;6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573;6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013;5,959,091; 5,942,664; 5,942,658, 5,880,275; 5,763,245; and 5,763,241),fungal disease resistance (U.S. Pat. Nos. 6,653,280; 6,573,361;6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436;6,316,407; and 6,506,962), virus resistance (U.S. Pat. Nos. 6,617,496;6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematoderesistance (U.S. Pat. No. 6,228,992), bacterial disease resistance (U.S.Pat. No. 5,516,671), plant growth and development (U.S. Pat. Nos.6,723,897 and 6,518,488), starch production (U.S. Pat. Nos. 6,538,181;6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils production(U.S. Pat. Nos. 6,444,876; 6,426,447; and 6,380,462), high oilproduction (U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; and6,476,295), modified fatty acid content (U.S. Pat. Nos. 6,828,475;6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767;6,537,750; 6,489,461; and 6,459,018), high protein production (U.S. Pat.No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhancedanimal and human nutrition (U.S. Pat. Nos. 6,723,837; 6,653,530;6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Pat. Nos.RE37,543; 6,228,623; and U.S. Pat. Nos. 5,958,745, and 6,946,588),environmental stress resistance (U.S. Pat. No. 6,072,103),pharmaceutical peptides and secretable peptides (U.S. Pat. Nos.6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processingtraits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No.6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzymeproduction (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No.6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seedproduction (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. Nos.6,576,818; 6,271,443; 5,981,834; and 5,869,720) and biofuel production(U.S. Pat. No. 5,998,700).

Alternatively, a gene of agronomic interest can affect the abovementioned plant characteristics or phenotypes by encoding a RNA moleculethat causes the targeted modulation of gene expression of an endogenousgene, for example by antisense (see, e.g. U.S. Pat. No. 5,107,065);inhibitory RNA (“RNAi,” including modulation of gene expression bymiRNA-, siRNA-, trans-acting siRNA-, and phased sRNA-mediatedmechanisms, e.g., as described in published applications U.S.2006/0200878 and U.S. 2008/0066206, and in U.S. patent application Ser.No. 11/974,469); or cosuppression-mediated mechanisms. The RNA couldalso be a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see,e.g., U.S. 2006/0200878) engineered to cleave a desired endogenous mRNAproduct. Methods are known in the art for constructing and introducingconstructs into a cell in such a manner that the transcribable DNAmolecule is transcribed into a molecule that is capable of causing genesuppression.

Selectable Markers

Selectable marker transgenes may also be used with the regulatoryelements of the invention. As used herein the term “selectable markertransgene” refers to any transcribable DNA molecule whose expression ina transgenic plant, tissue or cell, or lack thereof, can be screened foror scored in some way. Selectable marker genes, and their associatedselection and screening techniques, for use in the practice of theinvention are known in the art and include, but are not limited to,transcribable DNA molecules encoding ß-glucuronidase (GUS), luciferase,green fluorescent protein (GFP), proteins that confer antibioticresistance, and proteins that confer herbicide tolerance.

Cell Transformation

The invention is also directed to a method of producing transformedcells and plants that comprise one or more regulatory elements operablylinked to a transcribable DNA molecule.

The term “transformation” refers to the introduction of a DNA moleculeinto a recipient host. As used herein, the term “host” refers tobacteria, fungi, or plants, including any cells, tissues, organs, orprogeny of the bacteria, fungi, or plants. Plant tissues and cells ofparticular interest include protoplasts, calli, roots, tubers, seeds,stems, leaves, seedlings, embryos, and pollen.

As used herein, the term “transformed” refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is inherited by subsequent progeny. A“transgenic” or “transformed” cell or organism may also includes progenyof the cell or organism and progeny produced from a breeding programemploying such a transgenic organism as a parent in a cross andexhibiting an altered phenotype resulting from the presence of a foreignDNA molecule. The introduced DNA molecule may also be transientlyintroduced into the recipient cell such that the introduced DNA moleculeis not inherited by subsequent progeny. The term “transgenic” refers toa bacterium, fungus, or plant containing one or more heterologous DNAmolecules.

There are many methods well known to those of skill in the art forintroducing DNA molecules into plant cells. The process generallycomprises the steps of selecting a suitable host cell, transforming thehost cell with a vector, and obtaining the transformed host cell.Methods and materials for transforming plant cells by introducing aconstruct into a plant genome in the practice of this invention caninclude any of the well-known and demonstrated methods. Suitable methodsinclude, but are not limited to, bacterial infection (e.g.,Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., byPEG-mediated transformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, and accelerationof DNA coated particles), among others.

Host cells may be any cell or organism, such as a plant cell, algalcell, algae, fungal cell, fungi, bacterial cell, or insect cell. Inspecific embodiments, the host cells and transformed cells may includecells from crop plants.

A transgenic plant subsequently may be regenerated from a transgenicplant cell of the invention. Using conventional breeding techniques orself-pollination, seed may be produced from this transgenic plant. Suchseed, and the resulting progeny plant grown from such seed, will containthe recombinant DNA molecule of the invention, and therefore will betransgenic.

Transgenic plants of the invention can be self-pollinated to provideseed for homozygous transgenic plants of the invention (homozygous forthe recombinant DNA molecule) or crossed with non-transgenic plants ordifferent transgenic plants to provide seed for heterozygous transgenicplants of the invention (heterozygous for the recombinant DNA molecule).Both such homozygous and heterozygous transgenic plants are referred toherein as “progeny plants.” Progeny plants are transgenic plantsdescended from the original transgenic plant and containing therecombinant DNA molecule of the invention. Seeds produced using atransgenic plant of the invention can be harvested and used to growgenerations of transgenic plants, i.e., progeny plants of the invention,comprising the construct of this invention and expressing a gene ofagronomic interest. Descriptions of breeding methods that are commonlyused for different crops can be found in one of several reference books,see, e.g., Allard, Principles of Plant Breeding, John Wiley & Sons, NY,U. of CA, Davis, Calif., 50-98 (1960); Simmonds, Principles of CropImprovement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen,Plant breeding Perspectives, Wageningen (ed), Center for AgriculturalPublishing and Documentation (1979); Fehr, Soybeans: Improvement,Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr,Principles of Variety Development, Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376 (1987).

The transformed plants may be analyzed for the presence of the gene orgenes of interest and the expression level and/or profile conferred bythe regulatory elements of the invention. Those of skill in the art areaware of the numerous methods available for the analysis of transformedplants. For example, methods for plant analysis include, but are notlimited to, Southern blots or northern blots, PCR-based approaches,biochemical analyses, phenotypic screening methods, field evaluations,and immunodiagnostic assays. The expression of a transcribable DNAmolecule can be measured using TaqMan® (Applied Biosystems, Foster City,Calif.) reagents and methods as described by the manufacturer and PCRcycle times determined using the TaqMan® Testing Matrix. Alternatively,the Invader® (Third Wave Technologies, Madison, Wis.) reagents andmethods as described by the manufacturer can be used to evaluatetransgene expression.

The invention also provides for parts of a plant of the invention. Plantparts include, but are not limited to, leaves, stems, roots, tubers,seeds, endosperm, ovule, and pollen. Plant parts of the invention may beviable, nonviable, regenerable, and/or non-regenerable. The inventionalso includes and provides transformed plant cells comprising a DNAmolecule of the invention. The transformed or transgenic plant cells ofthe invention include regenerable and/or non-regenerable plant cells.

The invention also provides a commodity product that is produced from atransgenic plant or part thereof containing the recombinant DNA moleculeof the invention. Commodity products of the invention contain adetectable amount of DNA comprising a DNA sequence selected from thegroup consisting of SEQ ID NO:1-37. As used herein, a “commodityproduct” refers to any composition or product which is comprised ofmaterial derived from a transgenic plant, seed, plant cell, or plantpart containing the recombinant DNA molecule of the invention. Commodityproducts include but are not limited to processed seeds, grains, plantparts, and meal. A commodity product of the invention will contain adetectable amount of DNA corresponding to the recombinant DNA moleculeof the invention. Detection of one or more of this DNA in a sample maybe used for determining the content or the source of the commodityproduct. Any standard method of detection for DNA molecules may be used,including methods of detection disclosed herein.

The invention may be more readily understood through reference to thefollowing examples, which are provided by way of illustration, and arenot intended to be limiting of the invention, unless specified. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the following examples represent techniques discovered bythe inventors to function well in the practice of the invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention, thereforeall matter set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

EXAMPLES Example 1 Identification and Cloning of Regulatory Elements

Regulatory expression element groups (EXPs) and transcriptiontermination regions (3′ UTRs) were identified and cloned from thegenomic DNA of the dicot species Medicago truncatula (Barrel Medic). Theselection of the Medicago truncatula 3′ UTRs was, in part, based onexpression patterns observed in homologous soybean genes.

The identification and cloning of Medicago truncatula 3′ UTRs began withthe selection of soybean genes of interest based upon the soybean genes'expression pattern in soy tissue surveys and proprietary transcriptprofiling experiments. The selected soybean genes were then used to findhomologous genes in Medicago truncatula using publicly available DNAsequences. Next, tissue samples derived from Medicago truncatula wereisolated from plants grown under different environmental conditions.Then, messenger RNA (mRNA) was isolated from the Medicago tissues andused in real time polymerase chain reaction (PCR) experiments todetermine the expression pattern of the Medicago genes. From theseexperiments, a subset of the Medicago truncatula genome was selected forcloning and characterization.

Using public Medicago truncatula sequence data, a bioinformatic analysiswas performed to identify regulatory elements within the selectedMedicago gene loci. For example, bioinformatic analysis was performed toidentify 3′ UTR sequences that comprise the polyadenylation andtermination regions of the mRNA and sequences extending further to theend of the identified gene locus. Amplification primers were thendesigned and used to amplify each of the identified regulatory elementDNA fragments, such as 3′ UTR DNA fragments, DNA fragments comprising apromoter, leader and intron, and DNA fragments comprising a promoter andleader. The resulting DNA fragments were ligated into base plantexpression vectors and sequenced.

For applicable DNA fragments, an analysis of the regulatory elementtranscription start site (TSS) and intron/exon splice junctions was thenperformed using transformed plant protoplasts. In this analysis, theprotoplasts were transformed with the plant expression vectorscomprising the cloned DNA fragments operably linked to a heterologoustranscribable DNA molecule. Next, the 5′ RACE System for RapidAmplification of cDNA Ends, Version 2.0 (Invitrogen, Carlsbad, Calif.92008) was used to confirm the regulatory element TSS and intron/exonsplice junctions by analyzing the DNA sequence of the produced mRNAtranscripts.

The DNA sequences of the identified 3′ UTRs are provided herein as SEQID NOs: 1-30. In addition, identified promoter DNA sequences areprovided herein as SEQ ID NOs: 32 and 36; identified leader DNAsequences are provided herein as SEQ ID NOs: 33 and 37; and anidentified intron DNA sequence is provided as SEQ ID NO: 34. Further,the DNA sequences of the identified EXPs are provided herein as SEQ IDNOs: 31 and 35. The regulatory expression element group EXP-Mt.Ubq2:1:2(SEQ ID NO: 31) comprises a promoter element, P-Mt.Ubq2-1:1:1 (SEQ IDNO: 32), operably linked 5′ to a leader element, L-Mt.Ubq2-1:1:1 (SEQ IDNO: 33), operably linked 5′ to an intron element, I-Mt.Ubq2-1:1:2 (SEQID NO: 34) and the regulatory expression element groupEXP-Mt.AC145767v28:1:1 (SEQ ID NO: 35) comprises a promoter element,P-Mt.AC145767v28-1:2:1 (SEQ ID NO: 36), operably linked 5′ to a leaderelement, L-Mt.AC145767v28-1:1:2 (SEQ ID NO: 37). Each of the DNAsequences identified and cloned from Medicago truncatula are listed inTable 1.

TABLE 1 3′ UTRs, Regulatory expression element groups, promoters,leaders, and introns cloned from Medicago truncatula. SEQ Description IDNO: Annotation T-Mt.AC145767v28-1:1:2 1 AC145767.28T-Mt.AC140914v20-1:2:1 2 AC140914.20 T-Mt.AC139600v16-1:2:1 3AC139600.16 T-Mt.AC153125V10-1:2:1 4 AC153125.10 T-Mt.Apx-1:1:2 5cytosolic ascorbate peroxidase T-Mt.EF1a-1:1:2 6 elongation factor 1alpha T-Mt.Expr1-1:2:1 7 putative oxidoreductase T-Mt.FBA-1:1:5 8fructose biphasphate aldolase, cytoplasmic isozyme 2 T-Mt.FBA-1:2:1 9fructose biphasphate aldolase, cytoplasmic isozyme 2 T-Mt.Gapdh-1:2:1 10glyceraldehyde-3-phosphate dehydrogenase T-Mt.Gpi-1:2:1 11 GPI-anchoredprotein T-Mt.Hsp20-1:2:1 12 heat shock protein 20 T-Mt.Lhcb2-1:2:1 13chlorophyll a/b binding protein type II precursor T-Mt.Lox-1-1:2:1 14lipoxygenase T-Mt.Methm-1:2:1 155-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferaseT-Mt.MP21-1:2:1 16 seed maturation protein T-Mt.Oxr-1:2:1 17 putativeoxidoreductase T-Mt.Pip1-1:2:1 18 plasma membrane integral proteinT-Mt.Prx-1:1:1 19 peroxidase T-Mt.PSII-T_A-1:2:1 20 photosystem II 5 kDaprotein, chloroplast precursor T-Mt.PSII-T_B-1:2:1 21 photosystem II 5kDa protein, chloroplast precursor T-Mt.Pt1-1:2:2 22 phosphateTransporter T-Mt.Pt2-1:2:2 23 phosphate Transporter T-Mt.RD22-1:2:1 24dehydration-responsive protein T-Mt.RpL3-1:2:1 25 ribosomal protein L3T-Mt.Sali3-2-1:2:1 26 aluminum-induced Sali3-2 protein T-Mt.Scp-1:2:1 27serine carboxypeptidase-related protein T-Mt.SeqID#21-1:2:1 28peroxidase T-Mt.Sui1-1:1:2 29 SUI1 translation initiation factorT-Mt.Zfp-1:2:1 30 CCCH-type zinc finger protein EXP-Mt.Ubq2:1:2 31Ubiquitin 2 P-Mt.Ubq2-1:1:1 32 Ubiquitin 2 L-Mt.Ubq2-1:1:1 33 Ubiquitin2 I-Mt.Ubq2-1:1:2 34 Ubiquitin 2 EXP-Mt.AC145767v28:1:1 35 AC145767.28P-Mt.AC145767v28-1:2:1 36 AC145767.28 L-Mt.AC145767v28-1:1:2 37AC145767.28

Example 2 Analysis of the Effect of 3′ UTRs on Constitutive GUSExpression in Soybean Leaf Protoplasts

Soybean leaf protoplasts were transformed with vectors, specificallyplasmid constructs, to assess the effect of selected Medicago truncatula3′ UTRs on expression. Soybean leaf protoplasts were transformed withDNA vectors containing a constitutive EXP sequence driving expression ofthe β-glucuronidase (GUS) transgene operably linked to a Medicago 3′UTR. These Medicago 3′ UTR-transformed soybean leaf protoplasts werecompared to soybean leaf protoplast in which expression of the GUStransgene was driven by a constitutive promoter, and the GUS transgenewas operably linked to a 3′ UTR derived from Gossypium hirsutum orGossypium barbadense.

The plant vectors utilized in these experiments were built using cloningmethods known in the art. The resulting vectors comprised a left borderregion from A. tumefaciens; a first transgene expression cassette forselection of transformed plant cells that confers resistance to eitherthe herbicide glyphosate or the antibiotic spectinomycin (both driven bythe Arabidopsis Actin 7 promoter); a second transgene expressioncassette used to assess the activity of the 3′ UTR, which comprised anEXP or promoter sequence operably linked 5′ to a DNA sequence for GUSthat possesses a processable intron (GUS-2, SEQ ID NO: 44), which isoperably linked 5′ to 3′ UTR derived from Medicago truncatula, Gossypiumhirsutum, or Gossypium barbadense; and a right border region from A.tumefaciens. The vectors that comprised a 3′ UTR derived from Medicago(i.e., pMON109593, pMON116803, pMON116812, pMON116813, pMON116815,pMON116826, pMON116827, pMON116830, pMON122852, pMON122853, pMON122854,pMON122855, pMON122856, pMON122857, pMON122858, pMON122859, pMON122862,pMON122864, pMON122865, pMON122866, pMON122867, and pMON122868) used theconstitutive regulatory expression element groupEXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 42) to drive GUS. The vectorsthat comprised a 3′ UTR derived from Gossypium hirsutum or Gossypiumbarbadense (i.e., pMON81345, pMON81347, and pMON83002) used theconstitutive promoter P-CaMV.35S-enh-1:1:11 (SEQ ID NO: 43) to driveGUS.

Table 2 provides the plasmid constructs with the corresponding 3′ UTRand SEQ ID NO used to transform the soybean protoplasts in experimentspresented in this Example.

TABLE 2 Plasmid constructs used to transform soybean leaf protoplastsand 3′ UTR descriptions. Plasmid SEQ Construct 3′ UTR Description ID NO:pMON81345 T-Gb.FbL2-1:1:1 41 pMON81347 T-Gh.E6-4A-0:2:1 38 pMON83002T-Gb.H6-1:2:1 39 pMON109593 T-Mt.Pt1-1:2:2 22 pMON116803T-Mt.AC140914v20-1:2:1 2 pMON116812 T-Mt.Lhcb2-1:2:1 13 pMON116813T-Mt.PSII-T_B-1:2:1 21 pMON116815 T-Mt.AC145767v28-1:1:2 1 pMON116826T-Mt.Lox-1-1:2:1 14 pMON116827 T-Mt.Gpi-1:2:1 11 pMON116830T-Mt.Scp-1:2:1 27 pMON122852 T-Mt.Methm-1:2:1 15 pMON122853T-Mt.Prx-1:1:1 19 pMON122854 T-Mt.Gapdh-1:2:1 10 pMON122855T-Mt.FBA-1:1:5 8 pMON122856 T-Mt.Zfp-1:2:1 30 pMON122857T-Mt.AC139600v16-1:2:1 3 pMON122858 T-Mt.MP21-1:2:1 16 pMON122859T-Mt.Oxr-1:2:1 17 pMON122862 T-Mt.Sui1-1:1:2 29 pMON122864T-Mt.Pip1-1:2:1 18 pMON122865 T-Mt.AC153125V10-1:2:1 4 pMON122866T-Mt.Sali3-2-1:2:1 26 pMON122867 T-Mt.Hsp20-1:2:1 12 pMON122868T-Mt.Expr1-1:2:1 7

Two plant vectors, specifically plasmid constructs, for use inco-transformation and normalization of data were also built usingcloning methods known in the art. Each of these plasmid constructscontained a specific luciferase coding sequence that was driven by aconstitutive EXP. The plant vector pMON19437 comprised an expressioncassette with a constitutive EXP comprising a promoter operably linked5′ to a leader sequence which is operably linked 5′ to an intron(EXP-CaMV.35S-enh+Zm.DnaK:1:1, SEQ ID NO: 47), operably linked 5′ to afirefly (Photinus pyralis) luciferase coding sequence (LUCIFERASE:1:3,SEQ ID NO: 45), operably linked 5′ to a 3′ UTR from the Agrobacteriumtumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 49).The plant vector pMON63934 comprised an expression cassette with aconstitutive EXP sequence comprising a promoter operably linked 5′ to aleader sequence (EXP-CaMV.35S-enh-Lhcb1, SEQ ID NO: 48), operably linked5′ to a sea pansy (Renilla reniformis) luciferase coding sequence(CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO: 46), operably linked 5′ to a3′ UTR from the Agrobacterium tumefaciens nopaline synthase gene(T-AGRtu.nos-1:1:13, SEQ ID NO: 49).

The soybean leaf protoplasts were transformed using a polyethyleneglycol (PEG)-based transformation method, as is well known in the art.Each protoplast cell was transformed with the pMON19437 plasmidconstruct, the pMON63934 plasmid construct, and one of the plasmidconstructs presented in Table 2. After transformation, the transformedsoybean leaf protoplasts were incubated overnight in total darkness.Next, measurement of GUS and luciferase was conducted by placingaliquots of a lysed preparation of transformed cells into two differentsmall-well trays. One tray was used for GUS measurements, and a secondtray was used to perform a dual luciferase assay using the dualluciferase reporter assay system (Promega Corp., Madison, Wis.; see,e.g., Promega Notes Magazine, NO: 57, 1996, p.02).

One or two transformations were performed for each plasmid constructpresented in Table 2. The mean expression values for each 3′ UTR weredetermined from several samples from each transformation. Samplemeasurements were made using four replicates of each plasmid constructtransformation, or alternatively, three replicates of each plasmidconstruct per one of two transformation experiments. The mean GUS andluciferase expression levels are provided in Table 3. In this Table, thefirefly luciferase values (e.g., from expression of pMON19437) areprovided in the column labeled “FLuc” and the sea pansy luciferasevalues (e.g., from expression of pMON63934) are provided in the columnlabeled “RLuc.”

TABLE 3 Mean GUS and Luciferase assay values in transformed soybean leafprotoplasts. Plasmid SEQ Construct 3′ UTR Description ID NO: GUS FLucRLuc pMON81345 T-Gb.FbL2-1:1:1 41 795 2332.5 3701 pMON81347T-Gh.E6-4A-0:2:1 38 73 584.3 802 pMON83002 T-Gb.H6-1:2:1 39 91 1142.81995 pMON109593 T-Mt.Pt1-1:2:2 22 4783 3619 12341 pMON116803T-Mt.AC140914v20-1:2:1 2 15053 4801.7 15876 pMON116812 T-Mt.Lhcb2-1:2:113 9771 4202.3 10976 pMON116813 T-Mt.PSII-T_B-1:2:1 21 7482 3347.3 8395pMON116815 T-Mt.AC145767v28-1:1:2 1 30469 6428 17764 pMON116826T-Mt.Lox-1-1:2:1 14 22330 3580.5 9984 pMON116827 T-Mt.Gpi-1:2:1 11 269343.7 478 pMON116830 T-Mt.Scp-1:2:1 27 3909 4683.7 10180 pMON122852T-Mt.Methm-1:2:1 15 33403 11049 28226 pMON122853 T-Mt.Prx-1:1:1 19 1283311198 22722 pMON122854 T-Mt.Gapdh-1:2:1 10 14811 8775.5 25229 pMON122855T-Mt.FBA-1:1:5 8 40383 17826 50299 pMON122856 T-Mt.Zfp-1:2:1 30 2187016141.3 56362 pMON122857 T-Mt.AC139600v16-1:2:1 3 24386 6782.7 15024pMON122858 T-Mt.MP21-1:2:1 16 30753 12929.8 40571 pMON122859T-Mt.Oxr-1:2:1 17 14499 5586.7 15222 pMON122862 T-Mt.Sui1-1:1:2 29 2776814680 35263 pMON122864 T-Mt.Pip1-1:2:1 18 40579 15837.7 36515 pMON122865T-Mt.AC153125V10-1:2:1 4 34867 17285.5 52519 pMON122866T-Mt.Sali3-2-1:2:1 26 33664 11923 27663 pMON122867 T-Mt.Hsp20-1:2:1 127088 9885.3 19590 pMON122868 T-Mt.Expr1-1:2:1 7 14539 7563.5 22320

Further, to compare the relative activity of each 3′ UTR, GUS valueswere expressed as a ratio of GUS to luciferase activity and normalizedto the best expressing non-Medicago 3′ UTR, i.e., T-Gb.FbL2-1:1:1 (SEQID NO: 41). Table 4 shows the GUS/Luciferase ratios and the normalizedratios. Again, in this Table, the firefly luciferase values are labeled“FLuc” and the sea pansy luciferase values are labeled “RLuc.”

TABLE 4 GUS/FLuc and GUS/RLuc ratios of expression normalized withrespect to T- Gb.FbL2-1:1:1 (SEQ ID NO: 41) in transformed soybean leafprotoplasts. GUS/FLuc GUS/RLuc SEQ Normalized to Normalized to 3′ UTRDescription ID NO: GUS/FLuc GUS/RLuc T-Gb.FbL2-1:1:1 T-Gb.FbL2-1:1:1T-Gb.FbL2-1:1:1 41 0.34 0.21 1.00 1.00 T-Gh.E6-4A-0:2:1 38 0.12 0.090.37 0.42 T-Gb.H6-1:2:1 39 0.08 0.05 0.23 0.21 T-Mt.Pt1-1:2:2 22 1.320.39 3.88 1.80 T-Mt.AC140914v20-1:2:1 2 3.13 0.95 9.20 4.41T-Mt.Lhcb2-1:2:1 13 2.33 0.89 6.82 4.14 T-Mt.PSII-T_B-1:2:1 21 2.24 0.896.56 4.15 T-Mt.AC145767v28-1:1:2 1 4.74 1.72 13.91 7.98 T-Mt.Lox-1-1:2:114 6.24 2.24 18.30 10.41 T-Mt.Gpi-1:2:1 11 0.78 0.56 2.30 2.62T-Mt.Scp-1:2:1 27 0.83 0.38 2.45 1.79 T-Mt.Methm-1:2:1 15 3.02 1.18 8.875.51 T-Mt.Prx-1:1:1 19 1.15 0.56 3.36 2.63 T-Mt.Gapdh-1:2:1 10 1.69 0.594.95 2.73 T-Mt.FBA-1:1:5 8 2.27 0.80 6.65 3.74 T-Mt.Zfp-1:2:1 30 1.350.39 3.98 1.81 T-Mt.AC139600v16-1:2:1 3 3.60 1.62 10.55 7.56T-Mt.MP21-1:2:1 16 2.38 0.76 6.98 3.53 T-Mt.Oxr-1:2:1 17 2.60 0.95 7.614.43 T-Mt.Sui1-1:1:2 29 1.89 0.79 5.55 3.67 T-Mt.Pip1-1:2:1 18 2.56 1.117.52 5.17 T-Mt.AC153125V10-1:2:1 4 2.02 0.66 5.92 3.09T-Mt.Sali3-2-1:2:1 26 2.82 1.22 8.28 5.67 T-Mt.Hsp20-1:2:1 12 0.72 0.362.10 1.68 T-Mt.Expr1-1:2:1 7 1.92 0.65 5.64 3.03

As demonstrated in Table 4, GUS expression was enhanced using all of theselected Medicago 3′ UTRs compared to the 3′ UTRs derived from Gossypiumhirsutum or Gossypium barbadense. For example, expression of GUS was2.1- to 18.3-fold higher using a Medicago-derived 3′ UTR based upon theGUS/FLuc ratios normalized with respect to T-Gb.FbL2-1:1:1, the bestexpressing 3′ UTR of those derived from Gossypium hirsutum or Gossypiumbarbadense. Similarly, expression of GUS was 1.61- to 10.48-fold higherusing a Medicago-derived 3′ UTR based upon the GUS/RLuc ratiosnormalized with respect to T-Gb.FbL2-1:1:1.

Example 3 Analysis of the Effect of 3′ UTRs on Constitutive GUSExpression in Stably Transformed Soybean Plants

Soybean plants were transformed with vectors, specifically plasmidconstructs, to assess the effect of selected Medicago truncatula 3′ UTRson expression. Specifically, soybean plants were transformed withvectors containing a constitutive EXP sequence driving expression of theβ-glucuronidase (GUS) transgene operably linked to a Medicago 3′ UTR.These Medicago 3′ UTR-transformed soybean plants were compared totransformed soybean plants in which expression of the GUS transgene wasdriven by a constitutive promoter, and the GUS transgene was operablylinked to a 3′ UTR derived from Gossypium barbadense.

The plant vectors utilized in these experiments were built using cloningmethods known in the art. The resulting vectors comprised a left borderregion from A. tumefaciens; a first transgene expression cassette forselection of transformed plant cells that confers resistance to theantibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); asecond transgene expression cassette used to assess the activity of the3′ UTR, which comprised the regulatory expression element groupEXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 42) operably linked 5′ to acoding sequence for GUS that possesses a processable intron (GUS-2, SEQID NO: 44), which is operably linked 5′ to a 3′ UTR derived fromMedicago truncatula or Gossypium barbadense; and a right border regionfrom A. tumefaciens. The vectors that comprised a 3′ UTR derived fromMedicago were pMON109593, pMON116803, pMON116812, pMON116813,pMON116815, pMON116826, pMON116827, pMON116830, pMON122850, pMON122851,pMON122852, pMON122853, pMON122854, pMON122855, pMON122856, pMON122857,pMON122858, pMON122859, pMON122861, pMON122862, pMON122863, pMON122864,pMON122865, pMON122866, pMON122867, and pMON122868. The vector thatcomprised a 3′ UTR from Gossypium barbadense was pMON102167.

Table 5 provides the plasmid constructs with the corresponding 3′ UTRand SEQ ID NO used to transform the soybean plants in experimentspresented in this Example.

TABLE 5 Plasmid constructs used to transform soybean plants and the 3′UTR descriptions. Plasmid SEQ Construct 3′ UTR Description ID NO:pMON102167 T-Gb.E6-3b:1:1 40 pMON109593 T-Mt.Pt1-1:2:2 22 pMON116803T-Mt.AC140914v20-1:2:1 2 pMON116812 T-Mt.Lhcb2-1:2:1 13 pMON116813T-Mt.PSII-T_B-1:2:1 21 pMON116815 T-Mt.AC145767v28-1:1:2 1 pMON116826T-Mt.Lox-1-1:2:1 14 pMON116827 T-Mt.Gpi-1:2:1 11 pMON116830T-Mt.Scp-1:2:1 27 pMON122850 T-Mt.RpL3-1:2:1 25 pMON122851T-Mt.RD22-1:2:1 24 pMON122852 T-Mt.Methm-1:2:1 15 pMON122853T-Mt.Prx-1:1:1 19 pMON122854 T-Mt.Gapdh-1:2:1 10 pMON122855T-Mt.FBA-1:1:5 8 pMON122856 T-Mt.Zfp-1:2:1 30 pMON122857T-Mt.AC139600v16-1:2:1 3 pMON122858 T-Mt.MP21-1:2:1 16 pMON122859T-Mt.Oxr-1:2:1 17 pMON122861 T-Mt.Apx-1:1:2 5 pMON122862 T-Mt.Sui1-1:1:229 pMON122863 T-Mt.EF1a-1:1:2 6 pMON122864 T-Mt.Pip1-1:2:1 18 pMON122865T-Mt.AC153125V10-1:2:1 4 pMON122866 T-Mt.Sali3-2-1:2:1 26 pMON122867T-Mt.Hsp20-1:2:1 12 pMON122868 T-Mt.Expr1-1:2:1 7

The soybean plants were transformed using Agrobacterium-mediatedtransformation methods known in the art. Expression of GUS was assayedqualitatively using histological sections of selected tissues. For thehistochemical GUS analysis, whole tissue sections were incubated withthe GUS staining solution X-Gluc(5-bromo-4-chloro-3-indolyl-b-glucuronide) (1 mg/ml) for an appropriatelength of time, rinsed, and visually inspected for blue coloration. GUSactivity was qualitatively determined by direct visual inspection orinspection under a microscope using selected plant organs and tissues.The R₀ generation plants were inspected for expression in Vn5 Root, Vn5Sink Leaf, Vn5 Source Leaf, R1 Source Leaf, R1 Petiole, R1 Flower,Yellow Pod Embryo (approximately R8 development stage), Yellow PodCotyledon (approximately R8 development stage), R3 Immature Seed, R3Pod, and R5 Cotyledon.

The quantitative changes of GUS expression relative to expressionimparted by pMON102167, which comprised the 3′ UTR derived fromGossypium barbadense, was also analyzed, as demonstrated in Tables 6-13.For this quantitative analysis, total protein was extracted fromselected tissues of transformed plants. One microgram of total proteinwas used with the fluorogenic substrate4-methyleumbelliferyl-β-D-glucuronide (MUG) in a total reaction volumeof 50 μl. The reaction product, 4-methlyumbelliferone (4-MU), ismaximally fluorescent at high pH, where the hydroxyl group is ionized.Addition of a basic solution of sodium carbonate simultaneously stopsthe assay and adjusts the pH for quantifying the fluorescent product.Fluorescence was measured with excitation at 365 nm, emission at 445 nmusing a Fluoromax-3 (Horiba; Kyoto, Japan) with Micromax Reader, withslit width set at excitation 2 nm, emission 3 nm.

Tables 6 and 7 show the mean quantitative expression levels measured inthe R₀ generation plant tissues. Those tissues not assayed are shown asblank cells in both tables.

TABLE 6 Mean GUS expression in R₀ generation plants in Vn5 Root, Vn5Sink Leaf, Vn5 Source Leaf, R1 Source Leaf, R1 Petiole, and R1 Flower.Vn5 Vn5 R1 Plasmid SEQ Vn5 Sink Source Source R1 R1 Construct 3′ UTRDescription ID NO: Root Leaf Leaf Leaf Petiole Flower pMON102167T-Gb.E6-3b:1:1 40 400.90 551.61 605.29 350.93 412.30 pMON109593T-Mt.Pt1-1:2:2 22 740.77 654.50 946.25 579.76 342.11 215.37 pMON116803T-Mt.AC140914v20-1:2:1 2 1306.76 2269.95 2187.61 344.78 480.47 243.11pMON116812 T-Mt.Lhcb2-1:2:1 13 649.15 785.16 1103.30 644.76 297.30294.38 pMON116813 T-Mt.PSII-T B-1:2:1 21 382.80 891.91 1026.78 262.82253.94 179.31 pMON116815 T-Mt.AC145767v28-1:1:2 1 3817.28 1939.403250.38 1393.65 1001.37 876.08 pMON116826 T-Mt.Lox-1-1:2:1 14 1093.151626.41 2030.11 3315.25 1376.39 1980.93 pMON116827 T-Mt.Gpi-1:2:1 11839.31 1263.82 1172.16 617.58 457.17 235.01 pMON116830 T-Mt.Scp-1:2:1 27240.31 187.07 330.49 113.50 20.79 41.73 pMON122850 T-Mt.RpL3-1:2:1 25479.50 673.20 687.00 388.10 524.10 202.68 pMON122851 T-Mt.RD22-1:2:1 24897.98 287.52 667.63 325.50 1056.16 407.35 pMON122852 T-Mt.Methm-1:2:115 852.05 1003.70 456.38 883.30 560.70 184.02 pMON122853 T-Mt.Prx-1:1:119 858.88 591.51 362.40 841.82 459.48 220.29 pMON122854 T-Mt.Gapdh-1:2:110 957.90 910.53 343.90 583.62 570.15 198.11 pMON122855 T-Mt.FBA-1:1:5 81293.27 396.14 338.26 167.55 113.14 94.21 pMON122856 T-Mt.Zfp-1:2:1 30254.48 250.56 154.27 425.90 223.53 115.33 pMON122857T-Mt.AC139600v16-1:2:1 3 1035.43 1014.18 579.85 1631.94 921.34 421.81pMON122858 T-Mt.MP21-1:2:1 16 408.94 299.07 282.34 315.48 562.46 308.11pMON122859 T-Mt.Oxr-1:2:1 17 3228.98 1315.58 2092.77 849.69 406.58 98.10pMON122861 T-Mt.Apx-1:1:2 5 974.70 433.60 510.50 263.00 103.70 117.70pMON122862 T-Mt.Sui1-1:1:2 29 1131.24 710.62 604.88 342.22 182.58 219.67pMON122863 T-Mt.EF1a-1:1:2 6 667.00 281.00 398.30 171.40 323.10 281.30pMON122864 T-Mt.Pip1-1:2:1 18 448.00 203.00 240.00 401.00 369.00 355.00pMON122865 T-Mt.AC153125V10-1:2:1 4 385.42 160.51 298.16 239.01 104.6432.62 pMON122866 T-Mt.Sali3-2-1:2:1 26 2274.70 1176.10 1490.54 976.91753.02 45.26 pMON122867 T-Mt.Hsp20-1:2:1 12 753.94 544.73 395.30 675.68668.83 255.68 pMON122868 T-Mt.Expr1-1:2:1 7 1151.60 608.21 692.82 235.6287.40 157.45

TABLE 7 Mean GUS expression in R₀ generation plants in Yellow PodEmbryo, Yellow Pod Cotyledon, R3 Immature Seed, R3 Pod, and R5Cotyledon. Yellow Yellow R3 Plasmid SEQ Pod Pod Immature R3 R5 Construct3′ UTR Description ID NO: Embryo Cotyledon Seed Pod Cotyledon pMON102167T-Gb.E6-3b:1:1 40 47.86 49.45 67.45 433.54 101.34 pMON109593T-Mt.Pt1-1:2:2 22 18.56 170.11 28.63 406.13 71.91 pMON116803T-Mt.AC140914v20-1:2:1 2 100.42 181.62 209.92 467.72 190.51 pMON116812T-Mt.Lhcb2-1:2:1 13 74.53 120.30 163.76 526.08 407.40 pMON116813T-Mt.PSII-T B-1:2:1 21 127.65 279.84 78.12 282.34 50.92 pMON116815T-Mt.AC145767v28-1:1:2 1 358.03 1192.69 989.47 2309.72 566.93 pMON116826T-Mt.Lox-1-1:2:1 14 280.48 577.87 231.15 2868.17 341.60 pMON116827T-Mt.Gpi-1:2:1 11 118.18 127.74 10.96 37.22 27.80 pMON116830T-Mt.Scp-1:2:1 27 57.11 72.33 23.96 271.88 98.36 pMON122850T-Mt.RpL3-1:2:1 25 265.30 489.70 57.40 487.50 264.40 pMON122851T-Mt.RD22-1:2:1 24 95.88 189.41 121.12 1045.20 72.23 pMON122852T-Mt.Methm-1:2:1 15 153.46 320.64 53.24 686.92 518.51 pMON122853T-Mt.Prx-1:1:1 19 46.64 146.53 38.64 360.48 103.28 pMON122854T-Mt.Gapdh-1:2:1 10 165.11 160.40 66.44 464.75 245.85 pMON122855T-Mt.FBA-1:1:5 8 172.21 381.32 111.57 496.04 306.13 pMON122856T-Mt.Zfp-1:2:1 30 46.37 44.66 87.51 775.69 57.17 pMON122857T-Mt.AC139600v16-1:2:1 3 142.78 243.74 45.58 615.99 452.09 pMON122858T-Mt.MP21-1:2:1 16 102.11 260.98 137.76 667.18 169.16 pMON122859T-Mt.Oxr-1:2:1 17 192.92 539.13 74.44 950.85 43.69 pMON122861T-Mt.Apx-1:1:2 5 53.50 217.70 37.90 95.30 174.50 pMON122862T-Mt.Sui1-1:1:2 29 195.81 502.37 62.10 135.60 500.71 pMON122863T-Mt.EF1a-1:1:2 6 136.80 270.20 127.20 387.10 150.00 pMON122864T-Mt.Pip1-1:2:1 18 140.00 220.00 87.00 398.00 102.00 pMON122865T-Mt.AC153125V10-1:2:1 4 20.55 56.64 11.83 pMON122866 T-Mt.Sali3-2-1:2:126 126.53 334.27 59.33 pMON122867 T-Mt.Hsp20-1:2:1 12 136.36 242.5277.11 509.01 73.23 pMON122868 T-Mt.Expr1-1:2:1 7 201.21 186.14 208.371264.62 203.90

As demonstrated in Tables 6 and 7, expression driven by the same EXP wasdistinct in tissues of stably transformed soybean plants comprisingdifferent Medicago 3′ UTRs when compared to the Gossypiumbarbadense-derived 3′ UTR.

Tables 8 and 9 show the fold expression differences in the tissues ofstably transformed soybean plants comprising different Medicago 3′ UTRswhen compared to the Gossypium barbadense-derived 3′ UTR.

TABLE 8 Fold expression in R₀ generation transformed soybean plants inVn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 Source Leaf, R1 Petiole,and R1 Flower. Vn5 Vn5 R1 Plasmid SEQ Vn5 Sink Source Source R1Construct 3′ UTR Description ID NO: Root Leaf Leaf Leaf FlowerpMON102167 T-Gb.E6-3b:1:1 40 1.00 1.00 1.00 1.00 1.00 pMON109593T-Mt.Pt1-1:2:2 22 1.85 1.19 1.56 1.65 0.52 pMON116803T-Mt.AC140914v20-1:2:1 2 3.26 4.12 3.61 0.98 0.59 pMON116812T-Mt.Lhcb2-1:2:1 13 1.62 1.42 1.82 1.84 0.71 pMON116813 T-Mt.PSII-TB-1:2:1 21 0.95 1.62 1.70 0.75 0.43 pMON116815 T-Mt.AC145767v28-1:1:2 19.52 3.52 5.37 3.97 2.12 pMON116826 T-Mt.Lox-1-1:2:1 14 2.73 2.95 3.359.45 4.80 pMON116827 T-Mt.Gpi-1:2:1 11 2.09 2.29 1.94 1.76 0.57pMON116830 T-Mt.Scp-1:2:1 27 0.60 0.34 0.55 0.32 0.10 pMON122850T-Mt.RpL3-1:2:1 25 1.20 1.22 1.14 1.11 0.49 pMON122851 T-Mt.RD22-1:2:124 2.24 0.52 1.10 0.93 0.99 pMON122852 T-Mt.Methm-1:2:1 15 2.13 1.820.75 2.52 0.45 pMON122853 T-Mt.Prx-1:1:1 19 2.14 1.07 0.60 2.40 0.53pMON122854 T-Mt.Gapdh-1:2:1 10 2.39 1.65 0.57 1.66 0.48 pMON122855T-Mt.FBA-1:1:5 8 3.23 0.72 0.56 0.48 0.23 pMON122856 T-Mt.Zfp-1:2:1 300.63 0.45 0.25 1.21 0.28 pMON122857 T-Mt.AC139600v16-1:2:1 3 2.58 1.840.96 4.65 1.02 pMON122858 T-Mt.MP21-1:2:1 16 1.02 0.54 0.47 0.90 0.75pMON122859 T-Mt.Oxr-1:2:1 17 8.05 2.39 3.46 2.42 0.24 pMON122861T-Mt.Apx-1:1:2 5 2.43 0.79 0.84 0.75 0.29 pMON122862 T-Mt.Sui1-1:1:2 292.82 1.29 1.00 0.98 0.53 pMON122863 T-Mt.EF1a-1:1:2 6 1.66 0.51 0.660.49 0.68 pMON122864 T-Mt.Pip1-1:2:1 18 1.12 0.37 0.40 1.14 0.86pMON122865 T-Mt.AC153125V10-1:2:1 4 0.96 0.29 0.49 0.68 0.08 pMON122866T-Mt.Sali3-2-1:2:1 26 5.67 2.13 2.46 2.78 0.11 pMON122867T-Mt.Hsp20-1:2:1 12 1.88 0.99 0.65 1.93 0.62 pMON122868 T-Mt.Expr1-1:2:17 2.87 1.10 1.14 0.67 0.38

TABLE 9 Fold expression in R₀ generation transformed soybean plants inYellow Pod Embryo, Yellow Pod Cotyledon, R3 Immature Seed, R3 Pod, andR5 Cotyledon. Yellow Yellow R3 Plasmid SEQ Pod Pod Immature R3 R5Construct 3′ UTR Description ID NO: Embryo Cotyledon Seed Pod CotyledonpMON102167 T-Gb.E6-3b:1:1 40 1.00 1.00 1.00 1.00 1.00 pMON109593T-Mt.Pt1-1:2:2 22 0.39 3.44 0.42 0.94 0.71 pMON116803T-Mt.AC140914v20-1:2:1 2 2.10 3.67 3.11 1.08 1.88 pMON116812T-Mt.Lhcb2-1:2:1 13 1.56 2.43 2.43 1.21 4.02 pMON116813 T-Mt.PSII-TB-1:2:1 21 2.67 5.66 1.16 0.65 0.50 pMON116815 T-Mt.AC145767v28-1:1:2 17.48 24.12 14.67 5.33 5.59 pMON116826 T-Mt.Lox-1-1:2:1 14 5.86 11.693.43 6.62 3.37 pMON116827 T-Mt.Gpi-1:2:1 11 2.47 2.58 0.16 0.09 0.27pMON116830 T-Mt.Scp-1:2:1 27 1.19 1.46 0.36 0.63 0.97 pMON122850T-Mt.RpL3-1:2:1 25 5.54 9.90 0.85 1.12 2.61 pMON122851 T-Mt.RD22-1:2:124 2.00 3.83 1.80 2.41 0.71 pMON122852 T-Mt.Methm-1:2:1 15 3.21 6.480.79 1.58 5.12 pMON122853 T-Mt.Prx-1:1:1 19 0.97 2.96 0.57 0.83 1.02pMON122854 T-Mt.Gapdh-1:2:1 10 3.45 3.24 0.99 1.07 2.43 pMON122855T-Mt.FBA-1:1:5 8 3.60 7.71 1.65 1.14 3.02 pMON122856 T-Mt.Zfp-1:2:1 300.97 0.90 1.30 1.79 0.56 pMON122857 T-Mt.AC139600v16-1:2:1 3 2.98 4.930.68 1.42 4.46 pMON122858 T-Mt.MP21-1:2:1 16 2.13 5.28 2.04 1.54 1.67pMON122859 T-Mt.Oxr-1:2:1 17 4.03 10.90 1.10 2.19 0.43 pMON122861T-Mt.Apx-1:1:2 5 1.12 4.40 0.56 0.22 1.72 pMON122862 T-Mt.Sui1-1:1:2 294.09 10.16 0.92 0.31 4.94 pMON122863 T-Mt.EF1a-1:1:2 6 2.86 5.46 1.890.89 1.48 pMON122864 T-Mt.Pip1-1:2:1 18 2.93 4.45 1.29 0.92 1.01pMON122865 T-Mt.AC153125V10-1:2:1 4 0.43 1.15 0.12 pMON122866T-Mt.Sali3-2-1:2:1 26 2.64 6.76 0.59 pMON122867 T-Mt.Hsp20-1:2:1 12 2.854.90 1.14 1.17 0.72 pMON122868 T-Mt.Expr1-1:2:1 7 4.20 3.76 3.09 2.922.01

As demonstrated in Tables 8 and 9, expression in the tissues oftransformed soybean plants comprising different Medicago 3′ UTRs wasdistinct when compared to that of soybean plants transformed withpMON102167, which comprised a 3′ UTR derived from Gossypium barbadense.For example, two Medicago 3′ UTRs, T-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1)and T-Mt.Lox-1-1:2:1 (SEQ ID NO: 14) caused enhanced expression of theconstitutive EXP, EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 42), acrossall tissues. Other Medicago 3′ UTRs provided enhanced expression of theconstitutive EXP in some tissues, while reducing expression in others.For example, the 3′ UTR T-Mt.Sali3-2-1:2:1 (SEQ ID NO: 26) provided a2.19- to 8.05-fold increase in expression in the Vn5 Root, Vn5 SinkLeaf, Vn5 Source Leaf, R1 Source Leaf, Yellow Pod Embryo, and Yellow PodCotyledon, while reducing expression in the R1 Flower and R5 Cotyledon.Further, the 3′ UTR T-Mt.AC140914v20-1:2:1 (SEQ ID NO: 2) provided a1.88- to 4.12-fold increase in expression in Vn5 Root, Vn5 Sink Leaf,Vn5 Source Leaf, Yellow Pod Embryo, Yellow Pod Cotyledon, R3 ImmatureSeed, and R5 Cotyledon, while reducing expression in the R1 Source Leaf,R1 Flower, and keeping expression relatively the same in the R3 Pod. Inaddition, the 3′ UTR T-Mt.Oxr-1:2:1 (SEQ ID NO: 17) provided a 2.19-to10.90-fold increase in expression in Vn5 Root, Vn5 Sink Leaf, Vn5 SourceLeaf, R1 Source Leaf, Yellow Pod Embryo, Yellow Pod Cotyledon, and R3Pod, while reducing expression in the R1 Flower and R5 Cotyledon, andkeeping expression relatively the same in R3 Immature Seed.

Some of the transformed soybean plants comprising different Medicago 3′UTRs were taken to the R₁ generation. Tables 10 and 11 show the mean GUSexpression values of the assayed tissues. Tables 12 and 13 show the folddifference in expression relative to the 3′UTR derived from Gossypiumbarbadense.

TABLE 10 Mean GUS expression in R₁ generation transformed soybean plantsin Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 Source Leaf, R1 Petiole,and R1 Flower. Vn5 Vn5 R1 Plasmid SEQ Vn5 Sink Source Source R1 R1Construct 3′ UTR Description ID NO: Root Leaf Leaf Leaf Petiole FlowerpMON102167 T-Gb.E6-3b:1:1 40 934.22 992.31 1210.30 856.01 570.64 603.61pMON116813 T-Mt.PSII-T B-1:2:1 21 1462.92 1169.79 1495.65 1159.28 647.86506.70 pMON116815 T-Mt.AC145767v28-1:1:2 1 5555.77 5146.48 4447.422654.13 2825.41 2584.82 pMON122859 T-Mt.Oxr-1:2:1 17 3726.08 3090.413862.55 2666.68 1160.66 1041.40 pMON122866 T-Mt.Sali3-2-1:2:1 26 3438.352856.04 2510.49 2012.63 1087.69 919.57

TABLE 11 Mean GUS expression in R₁ generation transformed soybean plantsin Yellow Pod Embryo, Yellow Pod Cotyledon, R3 Immature Seed, R3 Pod andR5 Cotyledon. Yellow Yellow R3 Plasmid SEQ Pod Pod Immature R3 R5Construct 3′ UTR Description ID NO: Embryo Cotyledon Seed Pod CotyledonpMON102167 T-Gb.E6-3b:1:1 40 85.27 174.11 298.03 567.48 85.11 pMON116813T-Mt.PSII-T B-1:2:1 21 468.66 537.77 171.00 976.84 342.29 pMON116815T-Mt.AC145767v28-1:1:2 1 1314.44 2134.97 1039.30 4506.45 1842.61pMON122859 T-Mt.Oxr-1:2:1 17 730.81 1098.62 245.45 1947.45 423.40pMON122866 T-Mt.Sali3-2-1:2:1 26 686.08 988.27 488.62 1068.10 757.12

As demonstrated in Tables 10 and 11, expression driven by the same EXPwas distinct in tissues of stably transformed soybean plants comprisingdifferent Medicago 3′ UTRs when compared to the Gossypiumbarbadense-derived 3′ UTR. Tables 12 and 13 show the fold expressiondifferences in the tissues of stably transformed soybean plantscomprising different Medicago 3′ UTRs relative to tissues transformedwith pMON102167, which comprised a 3′ UTR derived from Gossypiumbarbadense.

TABLE 12 Fold expression differences in R₁ generation transformedsoybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 SourceLeaf, R1 Petiole, and R1. Vn5 Vn5 R1 Plasmid SEQ Vn5 Sink Source SourceR1 R1 Construct 3′ UTR Description ID NO: Root Leaf Leaf Leaf PetioleFlower pMON102167 T-Gb.E6-3b:1:1 40 1.00 1.00 1.00 1.00 1.00 1.00pMON116813 T-Mt.PSII-T B-1:2:1 21 1.57 1.18 1.24 1.35 1.14 0.84pMON116815 T-Mt.AC145767v28-1:1:2 1 5.95 5.19 3.67 3.10 4.95 4.28pMON122859 T-Mt.Oxr-1:2:1 17 3.99 3.11 3.19 3.12 2.03 1.73 pMON122866T-Mt.Sali3-2-1:2:1 26 3.68 2.88 2.07 2.35 1.91 1.52

TABLE 13 Fold expression differences in R₁ generation transformedsoybean plants in Yellow Pod Embryo, Yellow Pod Cotyledon, R3 ImmatureSeed, R3 Pod and R5 Cotyledon. Yellow Yellow R3 Plasmid SEQ Pod PodImmature R3 R5 Construct 3′ UTR Description ID NO: Embryo Cotyledon SeedPod Cotyledon pMON102167 T-Gb.E6-3b:1:1 40 1.00 1.00 1.00 1.00 1.00pMON116813 T-Mt.PSII-T B-1:2:1 21 5.50 3.09 0.57 1.72 4.02 pMON116815T-Mt.AC145767v28-1:1:2 1 15.42 12.26 3.49 7.94 21.65 pMON122859T-Mt.Oxr-1:2:1 17 8.57 6.31 0.82 3.43 4.97 pMON122866 T-Mt.Sali3-2-1:2:126 8.05 5.68 1.64 1.88 8.90

As demonstrated in Tables 12 and 13, several of the Medicago 3′ UTRsenhanced expression of the constitutive EXP element,EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 42), relative to plantstransformed with pMON102167, which comprised a 3′ UTR derived fromGossypium barbadense in the R₁ generation. For example, the 3′ UTRT-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1) provided a 3.10- to 21.65-foldenhancement of GUS expression in all of the tissues assayed. The 3′ UTRT-Mt.Sali3-2-1:2:1 (SEQ ID NO: 26) provided a 1.52- to 8.90-foldenhancement of GUS expression in all of the tissues assayed. The 3′ UTRT-Mt.Oxr-1:2:1 (SEQ ID NO: 17) provided enhancement in most tissues, butreduced expression in the R3 Immature Seed relative to plantstransformed with T-Gb.E6-3b:1:1 (SEQ ID NO: 40).

The forgoing experiments demonstrate that the Medicago truncatuladerived 3′ UTR elements affected expression of the constitutive EXPelement EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 42) in different waysdepending upon the specific 3′ UTR selected. In many cases, there was anenhancement of expression in certain tissues of plants transformed withplant expression vectors comprising a Medicago 3′ UTRs relative toplants transformed with pMON102167, which comprised a 3′ UTR derivedfrom Gossypium barbadense. However, the enhancement effect was not seenin all plant tissues and, in many cases, expression was attenuated insome tissues and enhanced in others using a Medicago 3′ UTR. Thus, theuse of selected Medicago 3′ UTRs allows for one to “fine tune” theexpression profile of a particular transgene and can be used incombination with different expression elements, such as promoters,leaders and introns, in operable linkage with a transcribable DNAmolecule to provide optimal expression in specific tissues, whilereducing expression in tissues that are less desirable for a specifictranscribable DNA molecule.

Example 4 Analysis of the Effect of 3′ UTRs on Seed Preferred GUSExpression in Stably Transformed Soybean Plants

Soybean plants were transformed with vectors, specifically plasmidconstructs, to assess the effect of selected Medicago 3′ UTRs onexpression. Specifically, soybean plants were transformed with DNAvectors containing a seed expressing EXP sequence driving expression ofthe β-glucuronidase (GUS) transgene operably linked to a Medicago 3′UTR. These Medicago 3′ UTR-transformed soybean plants were compared totransformed soybean plants in which expression of the GUS transgene wasdriven by a seed expressing EXP sequence and the GUS transgene wasoperably linked to 3′ UTR derived from Gossypium barbadense.

The plant vectors utilized in these experiments were built using cloningmethods known in the art. The resulting vectors comprised a left borderregion from A. tumefaciens; a first transgene expression cassette forselection of transformed plant cells that confers resistance to theantibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); asecond transgene expression cassette used to assess the activity of the3′ UTR, which comprised the EXP element, EXP-Gm.Sphas1:1:1 (SEQ ID NO:50), which provides seed preferred expression, operably linked 5′ to acoding sequence for GUS that possesses a processable intron (GUS-2, SEQID NO: 44), which is operably linked 5′ to a 3′ UTR derived fromMedicago truncatula or Gossypium barbadense; and a right border regionfrom A. tumefaciens. The plant expression vectors that comprised a 3′UTR derived from Medicago were pMON116832, pMON116834, pMON116835,pMON116841, pMON122869, pMON122870, pMON122871, pMON122872, pMON122873,pMON122874, pMON122875, pMON122876, pMON122878, pMON122879, pMON122880,pMON122881, pMON122882, pMON122883, pMON122885, pMON122887, pMON122888,and pMON126122. The vector that comprised a 3′ UTR from Gossypiumbarbadense was pMON83028.

Table 14 provides the plasmid constructs with the corresponding 3′ UTR,SEQ ID NO, and generation for which quantitative assay data is provided.

TABLE 14 Plasmid constructs used to transform soybean plants andcorresponding 3′ UTR. Generation For Plasmid SEQ which Data is Construct3′ UTR Description ID NO: Provided pMON83028 T-Gb.E6-3b:1:1 40 R₁pMON116832 T-Mt.AC140914v20-1:2:1 2 R₀ pMON116834 T-Mt.PSII-T_A-1:2:1 20R₀ pMON116835 T-Mt.AC145767v28-1:1:2 1 R₀ pMON116841 T-Mt.PSII-T_B-1:2:121 R₀ pMON122869 T-Mt.RpL3-1:2:1 25 R₀ pMON122870 T-Mt.RD22-1:2:1 24 R₀pMON122871 T-Mt.Methm-1:2:1 15 R₀ pMON122872 T-Mt.Prx-1:1:1 19 R₀pMON122873 T-Mt.Gapdh-1:2:1 10 R₀ pMON122874 T-Mt.FBA-1:2:1 9 R₀pMON122875 T-Mt.Zfp-1:2:1 30 R₀ and R₁ pMON122876 T-Mt.AC139600v16-1:2:13 R₀ pMON122878 T-Mt.Oxr-1:2:1 17 R₀ pMON122879 T-Mt.Apx-1:1:2 5 R₀ andR₁ pMON122880 T-Mt.Sui1-1:1:2 29 R₀ and R₁ pMON122881 T-Mt.EF1a-1:1:2 6R₀ and R₁ pMON122882 T-Mt.Pip1-1:2:1 18 R₀ pMON122883T-Mt.AC153125V10-1:2:1 4 R₀ pMON122885 T-Mt.Expr1-1:2:1 7 R₀ pMON122887T-Mt.Pt1-1:2:2 22 R₀ pMON122888 T-Mt.Pt2-1:2:2 23 R₀ pMON126122T-Mt.Expr1-1:2:1 7 R₀

The soybean plants were transformed and GUS assayed as described inExample 3. Tables 15 and 16 provide the quantitative mean GUS values forthe R₀ generation of stably transformed soybean plants.

TABLE 15 Mean GUS expression in R₀ generation of transformed soybeanplants in Yellow Pod Embryo, Yellow Pod Cotyledon, R3 Immature Seed, R3Pod, and R5 Cotyledon. Yellow Yellow R3 SEQ Pod Pod Immature R3 R5 3′UTRDescription ID NO: Embryo Cotyledon Seed Pod CotyledonT-Mt.AC140914v20-1:2:1 2 572 1045 9 6 8 T-Mt.PSII-T_A-1:2:1 20 210 371 76 61 T-Mt.AC145767v28-1:1:2 1 1445 4264 11 8 47 T-Mt.PSII-T_B-1:2:1 21218 774 15 16 60 T-Mt.RpL3-1:2:1 25 683 1087 T-Mt.RD22-1:2:1 24 31646809 30 15 24 T-Mt.Methm-1:2:1 15 459 2136 7 6 74 T-Mt.Prx-1:1:1 19 109794 9 6 42 T-Mt.Gapdh-1:2:1 10 241 745 6 5 T-Mt.FBA-1:2:1 9 622 772 10 6100 T-Mt.Zfp-1:2:1 30 192 193 2 2 31 T-Mt.AC139600v16-1:2:1 3 319 2150 86 157 T-Mt.Oxr-1:2:1 17 995 3220 5 4 235 T-Mt.Apx-1:1:2 5 41 272 10 9 10T-Mt.Sui1-1:1:2 29 120 546 15 116 16 T-Mt.EF1a-1:1:2 6 10 9 17T-Mt.Pip1-1:2:1 18 670 614 8 9 5 T-Mt.AC153125V10-1:2:1 4 2079 4192 8 662 T-Mt.Expr1-1:2:1 7 385 1092 11 5 299 T-Mt.Pt1-1:2:2 22 142 630 14 14426 T-Mt.Pt2-1:2:2 23 440 513 2 1 10 T-Mt.Expr1-1:2:1 7 527 1122 15 6154

TABLE 16 Mean GUS expression in R₀ generation transformed soybean plantsin Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 Source Leaf, R1 Petiole,and R1 Flower. Vn5 Vn5 R1 SEQ Vn5 Sink Source Source R1 R1 3′ UTRDescription ID NO: Root Leaf Leaf Leaf Petiole FlowerT-Mt.AC140914v20-1:2:1 2 23 4 6 4 4 4 T-Mt.PSII-T_A-1:2:1 20 29 5 8 6 33 T-Mt.AC145767v28-1:1:2 1 10 3 4 0 0 0 T-Mt.PSII-T_B-1:2:1 21 8 5 5 5 56 T-Mt.RpL3-1:2:1 25 60 26 22 7 8 9 T-Mt.RD22-1:2:1 24 21 2 3 12 11 11T-Mt.Methm-1:2:1 15 8 4 4 0 0 0 T-Mt.Prx-1:1:1 19 5 5 5 0 0 0T-Mt.Gapdh-1:2:1 10 20 8 6 8 6 8 T-Mt.FBA-1:2:1 9 9 3 3 18 15 17T-Mt.Zfp-1:2:1 30 41 13 14 7 5 6 T-Mt.AC139600v16-1:2:1 3 7 5 5 0 0 0T-Mt.Oxr-1:2:1 17 7 3 8 0 0 0 T-Mt.Apx-1:1:2 5 31 16 19 1173 294 357T-Mt.Sui1-1:1:2 29 29 20 19 10 5 4 T-Mt.EF1a-1:1:2 6 8 3 3 16 19 19T-Mt.Pip1-1:2:1 18 15 7 6 8 4 3 T-Mt.AC153125V10-1:2:1 4 16 5 3 0 0 0T-Mt.Expr1-1:2:1 7 22 8 10 6 3 3 T-Mt.Pt1-1:2:2 22 8 6 5 5 6 6T-Mt.Pt2-1:2:2 23 34 11 11 6 6 6 T-Mt.Expr1-1:2:1 7 15 6 8 5 4 4

As can be seen in Tables 15 and 16, most of the Medicago 3′ UTRsaffected expression of the seed preferred EXP element, EXP-Gm.Sphas1:1:1(SEQ ID NO: 50), in only seed-derived tissues, with the exception ofT-Mt.Apx-1:1:2 (SEQ ID NO: 5), which enhanced expression of GUS in theR1 Source Leaf, R1 Petiole, and R1 Flower. Several Medicago 3′ UTRsprovided high expression in the Yellow Pod Embryo and Yellow PodCotyledon, such as T-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1),T-Mt.RD22-1:2:1 (SEQ ID NO: 24), and T-Mt.AC153125V10-1:2:1 (SEQ ID NO:4). Thus, these 3′ UTRs may be ideal to enhance expression of a seedpromoter during the later stages of seed development. The 3′ UTRT-Mt.Expr1-1:2:1 (SEQ ID NO: 7) provided high expression in both R5Cotyledon and Yellow Pod Cotyledon relative to many of the other 3′UTRs, and thus may be useful in providing high cotyledon expression fora wider window of seed development. In some cases, the 3′ UTR provided amore uniform level of seed expression both in the Yellow Pod Embryo andYellow Pod Cotyledon, such as when T-Mt.FBA-1:2:1 (SEQ ID NO: 9),T-Mt.Zfp-1:2:1 (SEQ ID NO: 30), T-Mt.Pip1-1:2:1 (SEQ ID NO: 18), andT-Mt.Pt2-1:2:2 (SEQ ID NO: 23) were used.

The R₀ generation plants comprising T-Mt.Zfp-1:2:1 (SEQ ID NO: 30),T-Mt.Apx-1:1:2 (SEQ ID NO: 5), T-Mt.Sui1-1:1:2 (SEQ ID NO: 29), andT-Mt.EF1a-1:1:2 (SEQ ID NO: 6) were allowed to set seed and were plantedfor R₁ generation studies. Table 17 shows a comparison of the meanquantitative assay data for events comprising these R₁ generation plantscomprising Medicago 3′ UTRs and plants transformed with pMON83028, whichcomprised the 3′ UTR T-Gb.E6-3b:1:1 (SEQ ID NO: 40) derived fromGossypium barbadense.

TABLE 17 Mean GUS expression in R₁ generation transformed soybean plantsin Yellow Pod Embryo, Yellow Pod Cotyledon, and R5 Cotyledon. Plasmid 3′UTR SEQ Yellow Pod Yellow Pod R5 Construct Description ID NO: EmbryoCotyledon Cotyledon pMON83028 T-Gb.E6-3b:1:1 40 102 362 7 pMON122875T-Mt.Zfp-1:2:1 30 56 153 498 pMON122879 T-Mt.Apx-1:1:2 5 205 645 777pMON122880 T-Mt.Sui1-1:1:2 29 462 1241 355 pMON122881 T-Mt.EF1a-1:1:2 6415 1059 11

As can be seen in Table 17, the Medicago 3′ UTRs affected expressiondifferently than T-Gb.E6-3b:1:1 in the embryo and cotyledon tissues. Forexample, T-Mt.Apx-1:1:2 (SEQ ID NO: 5) and T-Mt.Sui1-1:1:2 (SEQ ID NO:29) enhanced expression of the seed-preferred EXP element in the YellowPod Embryo, Yellow Pod Cotyledon, and R5 Cotyledon relative toT-Gb.E6-3b:1:1. T-Mt.EF1a-1:1:2 (SEQ ID NO: 6) enhanced expression inthe Yellow Pod Embryo and Yellow Pod Cotyledon, but not in the R5Cotyledon. T-Mt.Zfp-1:2:1 (SEQ ID NO: 30) reduced expression in thelater developing Yellow Pod Embryo and Yellow Pod Cotyledon, butenhanced expression in the R5 Cotyledon.

Thus, each of the different Medicago 3′ UTRs affect expressiondifferentially in the developing seed when in operable linkage with aseed preferred promoter. These differences in the effect on expressioncan be utilized to provide a more refined and tailored approach to seedexpression and may be ideally suited for “fine tuning” the expressionprofile of specific transcribable DNA molecules where seed expression isdesired.

Example 5 Analysis of the Effect of 3′ UTRs on Constitutive GUSExpression in Stably Transformed Soybean Plants

Soybean plants were transformed with vectors, specifically plasmidconstructs, to assess the effect of selected Medicago truncatula 3′ UTRson expression. Specifically, soybean plants were transformed withvectors containing two different EXP elements that exhibit aconstitutive expression profile driving expression of theβ-glucuronidase (GUS) transgene operably linked to a Medicago 3′ UTR.These Medicago 3′ UTR-transformed plants were compared to transformedsoybean plants in which expression of the GUS transgene was operablylinked to a 3′ UTR derived from Gossypium barbadense.

The plant vectors utilized in these experiments were built using cloningmethods known in the art. The resulting vectors comprised a left borderregion from A. tumefaciens; a first transgene expression cassette forselection of transformed plant cells that confers resistance to theantibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); asecond transgene expression cassette used to assess the activity of the3′ UTR, which comprised the EXP elements EXP-CaMV.35S-enh+Ph.DnaK:1:3(SEQ ID NO: 42) or EXP-DaMV.FLT:1:2 (SEQ ID NO: 51) operably linked 5′to a coding sequence for GUS that possesses a processable intron (GUS-2,SEQ ID NO: 44), which is operably linked 5′ to a 3′ UTR derived fromMedicago truncatula or Gossypium barbadense; and a right border regionfrom A. tumefaciens. The vectors that comprised a 3′ UTR derived fromMedicago were pMON118768, pMON153701 and pMON116803. The vectors thatcomprised a 3′ UTR from Gossypium barbadense were pMON121042 andpMON102167.

Table 18 provides the plasmid constructs with the corresponding EXPelement, 3′ UTR and SEQ ID NO used to transform the soybean plantspresented in this Example.

TABLE 18 Plasmid constructs used to transform soybean plants and thecorresponding EXP element and 3′ UTR. 3′ Plasmid EXP SEQ UTR SEQConstruct EXP Description ID NO: 3′ UTR Description ID NO: pMON121042EXP-DaMV.FLT:1:2 51 T-Gb.E6-3b:1:1 40 pMON118768 EXP-DaMV.FLT:1:2 51T-Mt.Sali3-2-1:2:1 26 pMON153701 EXP-DaMV.FLT:1:2 51T-Mt.AC140914v20-1:2:1 2 pMON102167 EXP-CaMV.35S-enh + Ph.DnaK:1:3 42T-Gb.E6-3b:1:1 40 pMON122866 EXP-CaMV.35S-enh + Ph.DnaK:1:3 42T-Mt.Sali3-2-1:2:1 26 pMON116803 EXP-CaMV.35S-enh + Ph.DnaK:1:3 42T-Mt.AC140914v20-1:2:1 2

Plants were transformed and GUS assayed as described in Example 3.Tables 19 and 20 provide the quantitative mean GUS values for the R₀generation of stably transformed soybean plants.

TABLE 19 Mean GUS expression in R₀ generation transformed soybean plantsin Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 Source Leaf, R1 Petiole,and R1 Flower. Vn5 Vn5 R1 Vn5 Sink Source Source R1 R1 EXP Description3′ UTR Description Root Leaf Leaf Leaf Petiole Flower EXP-DaMV.FLT:1:2T-Gb.E6-3b:1:1 780.79 688.93 509.35 320.02 379.69 467.94EXP-DaMV.FLT:1:2 T-Mt.Sali3-2-1:2:1 4782.43 1009.59 1208.48 363.551425.76 1398.80 EXP-DaMV.FLT:1:2 T-Mt.AC140914v20-1:2:1 3792.66 725.381106.9 1831.99 4792.28 739.97 EXP-CaMV.35S-enh+Ph.DnaK:1:3T-Gb.E6-3b:1:1 400.90 551.61 605.29 350.93 412.30EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.Sali3-2-1:2:1 2274.70 1176.10 1490.54976.91 753.02 45.26 EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.AC140914v20-1:2:11306.76 2269.95 2187.61 344.78 480.47 243.11

TABLE 20 Mean GUS expression in R₀ generation transformed soybean plantsin Yellow Pod Embryo, Yellow Pod Cotyledon, R3 Immature Seed, R3 Pod andR5 Cotyledon. Yellow Yellow R3 Pod Pod Immature R3 R5 EXP Description 3′UTR Description Embryo Cotyledon Seed Pod Cotyledon EXP-DaMV.FLT:1:2T-Gb.E6-3b:1:1 104.58 115.16 340.02 859.14 64.18 EXP-DaMV.FLT:1:2T-Mt.Sali3-2-1:2:1 1582.51 832.99 84.88 1157.18 247.75 EXP-DaMV.FLT:1:2T-Mt.AC140914v20-1:2:1 961.14 1050.82 456.55 2455.53 861.1EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Gb.E6-3b:1:1 47.86 49.45 67.45 433.54101.34 EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.Sali3-2-1:2:1 126.53 334.2759.33 EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.AC140914v20-1:2:1 100.42 181.62209.92 467.72 190.51

As demonstrated in Tables 19 and 20, the Medicago 3′ UTRsT-Mt.Sali3-2-1:2:1 (SEQ ID NO: 26) and T-Mt.AC140914v20-1:2:1 (SEQ IDNO: 2) affected expression of the constitutive EXP elementEXP-DaMV.FLT:1:2 (SEQ ID NO: 51) differently than the Gossypiumbarbadense-derived 3′ UTR T-Gb.E6-3b:1:1 (SEQ ID NO: 40). In many of thesampled tissues, there was an enhancement of expression using theMedicago 3′ UTRs. With respect to the 3′ UTR T-Mt.AC140914v20-1:2:1,enhancement was seen in most tissues in plants also comprising the EXPelement EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 42). Tables 21 and 22show the fold differences of the quantitative GUS expression relative tothe expression imparted by pMON121042 (T-Gb.E6-3b:1:1 (SEQ ID NO: 40)),which comprises a 3′ UTR derived from Gossypium barbadense.

TABLE 21 Fold expression differences in R₁ generation transformedsoybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 SourceLeaf, R1 Petiole and R1 Flower. Vn5 Vn5 R1 Vn5 Sink Source Source R1 R1EXP Description 3′ UTR Description Root Leaf Leaf Leaf Petiole FlowerEXP-DaMV.FLT:1:2 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00 1.00EXP-DaMV.FLT:1:2 T-Mt.Sali3-2-1:2:1 6.13 1.47 2.37 1.14 3.76 2.99EXP-DaMV.FLT:1:2 T-Mt.AC140914v20-1:2:1 4.86 1.05 2.17 5.72 12.62 1.58EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.Sali3-2-1:2:1 5.67 2.13 2.46 2.78 0.11EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.AC140914v20-1:2:1 3.26 4.12 3.61 0.980.59

TABLE 22 Fold expression differences in R₁ generation transformedsoybean plants in Yellow Pod Embryo, Yellow Pod Cotyledon, R3 ImmatureSeed, R3 Pod and R5 Cotyledon. Yellow Yellow R3 Pod Pod Immature R3 R5EXP Description 3′ UTR Description Embryo Cotyledon Seed Pod CotyledonEXP-DaMV.FLT:1:2 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00EXP-DaMV.FLT:1:2 T-Mt.Sali3-2-1:2:1 15.13 7.23 0.25 1.35 3.86EXP-DaMV.FLT:1:2 T-Mt.AC140914v20-1:2:1 9.19 9.13 1.34 2.86 13.42EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.Sali3-2-1:2:1 2.64 6.76 0.59EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.AC140914v20-1:2:1 2.10 3.67 3.11 1.081.88

The forgoing experiments demonstrate that each of the Medicago 3′ UTRshas different effects upon the level of expression of each of theconstitutive EXP elements relative to pMON121042 (T-Gb.E6-3b:1:1 (SEQ IDNO: 40)), which comprises the 3′ UTR derived from Gossypium barbadense.For example, expression of EXP-DaMV.FLT:1:2 was enhanced 1.14-to15.13-fold in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 Source Leaf,R1 Petiole, R1 Flower, Yellow Pod Embryo, Yellow Pod Cotyledon, R3 Pod,and R5 Cotyledon, but reduced in the R3 Immature Seed usingT-Mt.Sali3-2-1:2:1. This same EXP element, when combined withT-Mt.AC140914v20-1:2:1, resulted in a 1.34- to 13.42-fold enhancement inVn5 Root, Vn5 Source Leaf, R1 Source Leaf, R1 Petiole, R1 Flower, YellowPod Embryo, Yellow Pod Cotyledon, R3 Immature Seed, R3 Pod, and R5Cotyledon, but remained about the same as T-Gb.E6-3b:1:1 (SEQ ID NO: 40)in the V5 Sink Leaf. Expression in Yellow Pod Embryo was about twicethat of Yellow Pod Cotyledon using T-Mt.Sali3-2-1:2:1 (15.13- vs.7.23-fold enhancement), while expression in these two tissues wasrelatively the same when using T-Mt.AC140914v20-1:2:1 (9.19- vs.9.13-fold enhancement). With respect to the EXP elementEXP-CaMV.35S-enh+Ph.DnaK:1:3, combination with T-Mt.AC140914v20-1:2:1produced less enhancement in many of the sampled tissues than when thissame 3′ UTR was combined with EXP-DaMV.FLT:1:2. In R1 Flower, there wasa reduction of expression relative to T-Gb.E6-3b:1:1 whenEXP-CaMV.35S-enh+Ph.DnaK:1:3 was combined with T-Mt.AC140914v20-1:2:1.The combination of EXP-CaMV.35S-enh+Ph.DnaK:1:3 with T-Mt.Sali3-2-1:2:1provided enhancement in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1Source Leaf, Yellow Pod Embryo, and Yellow Pod Cotyledon, but reducedexpression in the R1 Flower and R5 Cotyledon relative to T-Gb.E6-3b:1:1(SEQ ID NO: 40).

Each of the two Medicago 3′ UTRs, T-Mt.Sali3-2-1:2:1 andT-Mt.AC140914v20-1:2:1, affected the expression of the two differentconstitutive EXP elements, EXP-DaMV.FLT:1:2 andEXP-CaMV.35S-enh+Ph.DnaK:1:3, differently. In many tissues, there was anenhancement of expression relative to T-Gb.E6-3b:1:1 (SEQ ID NO: 40),but in some tissues, a reduction of expression occurred. Thus, by usingdifferent Medicago 3′ UTRs, one may be able to more precisely controlexpression in the plant and better “fine tune” the expression ofspecific transcribable DNA molecules to provide optimal expression wherethe expression of the transcribable DNA molecule is required, whilereducing expression in tissues that might negatively affect the plant.

Example 6 The Medicago truncatula 3′ UTR T-Mt.AC145767v28-1:1:2 CausesEnhancement of GUS Expression when Combined with Many Different EXPElements in Stably Transformed Soybean Plants

Soybean plants were transformed with vectors, specifically plasmidconstructs, to assess the effect of the Medicago 3′ UTRT-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1) on expression. Specifically, thesoybean plants were transformed with vectors containing severaldifferent EXPs with a constitutive expression profile driving expressionof the ß-glucuronidase (GUS) transgene operably linked to the Medicago3′ UTR T-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1). These Medicago 3′UTR-transformed soybean plants were compared to transformed soybeanplants in which the GUS transgene was operably linked to a 3′ UTRderived from Gossypium barbadense.

The vectors utilized in these experiments were built using cloningmethods known in the art. The resulting vectors comprised a left borderregion from A. tumefaciens; a first transgene expression cassette forselection of transformed plant cells that confers resistance to theantibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); asecond transgene expression cassette used to assess the activity of the3′ UTR T-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1) which comprises the EXPelements, EXP-Mt.AC145767v28:1:1 (SEQ ID NO: 35),EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 42), EXP-BSAcVNV.FLT:1:2 (SEQID NO: 52), EXP-CERV.FLT:1:2 (SEQ ID NO: 53), EXP-DaMV.FLT:1:2 (SEQ IDNO: 51), EXP-CUCme.eEF1a:1:1 (SEQ ID NO: 54), or EXP-Mt.Ubq2:1:2 (SEQ IDNO: 31) operably linked 5′ to a coding sequence for GUS that possesses aprocessable intron (GUS-2, SEQ ID NO: 44) which is operably linked 5′ tothe 3′ UTR T-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1) derived from Medicagotruncatula, or to the 3′UTRs T-Gb.E6-3b:1:1 (SEQ ID NO: 40) orT-Gb.FbL2-1:1:1 (SEQ ID NO: 41) derived from Gossypium barbadense; and aright border region from A. tumefaciens. The vectors that comprisedT-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1) were pMON118798, pMON116815,pMON118769, pMON153709, pMON118771, pMON153707, and pMON155502. Notably,vector pMON118798 comprised the native EXP-Mt.AC145767v28:1:1 which iscomprised of a promoter element operably linked to a leader elementcloned from the same gene locus as the 3′ UTR T-Mt.AC145767v28-1:1:2(SEQ ID NO: 1). The vectors that comprised the 3′UTR from Gossypiumbarbadense were pMON102167, pMON113874, pMON121030, pMON121042,pMON140827, and pMON125841.

Table 23 provides the plasmid constructs with the corresponding EXPelement, 3′ UTR, and SEQ ID NO used to transform the soybean plantspresented in this Example.

TABLE 23 Plasmid constructs used to transform soybean plants and thecorresponding EXP element and 3′ UTR. 3′ Plasmid EXP SEQ UTR SEQConstruct EXP Description ID NO: 3′ UTR Description ID NO: pMON118798EXP-Mt.AC145767v28:1:1 35 T-Mt.AC145767v28-1:1:2 1 pMON102167EXP-CaMV.35S-enh + Ph.DnaK:1:3 42 T-Gb.E6-3b:1:1 40 pMON116815EXP-CaMV.35S-enh + Ph.DnaK:1:3 42 T-Mt.AC145767v28-1:1:2 1 pMON113874EXP-BSAcVNV.FLT:1:2 52 T-Gb.E6-3b:1:1 40 pMON118769 EXP-BSAcVNV.FLT:1:252 T-Mt.AC145767v28-1:1:2 1 pMON121030 EXP-CERV.FLT:1:2 53T-Gb.E6-3b:1:1 40 pMON153709 EXP-CERV.FLT:1:2 53 T-Mt.AC145767v28-1:1:21 pMON121042 EXP-DaMV.FLT:1:2 51 T-Gb.E6-3b:1:1 40 pMON118771EXP-DaMV.FLT:1:2 51 T-Mt.AC145767v28-1:1:2 1 pMON140827EXP-CUCme.eEF1a:1:1 54 T-Gb.FbL2-1:1:1 41 pMON153707 EXP-CUCme.eEF1a:1:154 T-Mt.AC145767v28-1:1:2 1 pMON125841 EXP-Mt.Ubq2:1:2 31T-Gb.FbL2-1:1:1 41 pMON155502 EXP-Mt.Ubq2:1:2 31 T-Mt.AC145767v28-1:1:21

The soybean plants were transformed and GUS assayed as described inExample 3. Tables 24 and 25 provide the quantitative mean GUS values forthe R₀ generation of stably transformed soybean plants. Table cellsmarked as “bdl” indicate tissues that were quantitatively analyzed butin which expression was below the level of detection. Tables 26 and 27provide the fold changes in expression of each EXP element operablylinked to T-Mt.AC145767v28-1:1:2 relative to T-Gb.E6-3b:1:1(SEQ ID NO:40).

TABLE 24 Mean GUS expression in R₀ generation transformed soybean plantsin Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 Source Leaf, R1 Petioleand R1 Flower. Vn5 Vn5 R1 Vn5 R1 Sink Source Source R1 R1 EXPDescription 3′ UTR Description Root Root Leaf Leaf Leaf Petiole FlowerEXP-Mt.AC145767v28:1:1 T-Mt.AC145767v28-1:1:2 59.00 71.00 32.00 34.0033.00 23.00 bdl EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Gb.E6-3b:1:1 400.90618.03 551.61 605.29 350.93 412.30 EXP-CaMV.35S-enh+Ph.DnaK:1:3T-Mt.AC145767v28-1:1:2 3817.28 1939.40 3250.38 1393.65 1001.37 876.08EXP-BSAcVNV.FLT:1:2 T-Gb.E6-3b:1:1 111.12 19.96 19.46 17.47 88.14 64.38EXP-BSAcVNV.FLT:1:2 T-Mt.AC145767v28-1:1:2 6514.58 1081.72 477.98 419.52227.72 1380.90 581.97 EXP-CERV.FLT:1:2 T-Gb.E6-3b:1:1 378.02 344.15480.25 177.64 285.15 130.87 EXP-CERV.FLT:1:2 T-Mt.AC145767v28-1:1:26711.26 1618.72 3262.73 2995.09 5071.90 3608.75 EXP-DaMV.FLT:1:2T-Gb.E6-3b:1:1 780.79 688.93 509.35 320.02 379.69 467.94EXP-DaMV.FLT:1:2 T-Mt.AC145767v28-1:1:2 9322.50 3655.79 5870.15 3923.472313.08 3610.84 2131.16 EXP-CUCme.eEF1a:1:1 T-Gb.FbL2-1:1:1 189.24153.52 59.60 37.44 103.01 130.60 130.38 EXP-CUCme.eEF1a:1:1T-Mt.AC145767v28-1:1:2 2300.06 160.99 216.21 744.44 1628.65 405.97EXP-Mt.Ubq2:1:2 T-Gb.FbL2-1:1:1 800.93 202.73 275.48 143.60 1195.97482.13 EXP-Mt.Ubq2:1:2 T-Mt.AC145767v28-1:1:2 855.00 293.68 1118.76254.25 875.67 398.10

TABLE 25 Mean GUS expression in R₀ generation transformed soybean plantsin Yellow Pod Embryo, Yellow Pod Cotyledon, R3 Immature Seed, R3 Pod andR5 Cotyledon. Yellow Yellow R3 Pod Pod Immature R3 R5 EXP Description 3′UTR Description Embryo Cotyledon Seed Pod CotyledonEXP-Mt.AC145767v28:1:1 T-Mt.AC145767v28-1:1:2 31.00 27.00 bdl bdl 26.00EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Gb.E6-3b:1:1 47.86 49.45 67.45 433.54101.34 EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.AC145767v28-1:1:2 358.031192.69 989.47 2309.72 566.93 EXP-BSAcVNV.FLT:1:2 T-Gb.E6-3b:1:1 28.3162.63 24.08 115.00 11.35 EXP-BSAcVNV.FLT:1:2 T-Mt.AC145767v28-1:1:2547.47 207.69 128.15 927.48 65.67 EXP-CERV.FLT:1:2 T-Gb.E6-3b:1:1 68.5770.12 64.42 264.62 34.43 EXP-CERV.FLT:1:2 T-Mt.AC145767v28-1:1:2 1474.354242.09 2441.01 7209.69 900.82 EXP-DaMV.FLT:1:2 T-Gb.E6-3b:1:1 104.58115.16 340.02 859.14 64.18 EXP-DaMV.FLT:1:2 T-Mt.AC145767v28-1:1:22806.65 1814.87 518.90 3720.59 401.66 EXP-CUCme.eEF1a:1:1T-Gb.FbL2-1:1:1 200.28 291.26 58.21 131.17 114.29 EXP-CUCme.eEF1a:1:1T-Mt.AC145767v28-1:1:2 1029.69 1883.48 209.77 1122.51 521.64EXP-Mt.Ubq2:1:2 T-Gb.FbL2-1:1:1 129.84 83.45 400.15 875.75 72.66EXP-Mt.Ubq2:1:2 T-Mt.AC145767v28-1:1:2 247.18 1324.98 352.81

TABLE 26 Fold expression differences in R₁ generation transformedsoybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, R1 SourceLeaf, R1 Petiole and R1 Flower. Vn5 Vn5 R1 Vn5 Sink Source Source R1 R1EXP Description 3′ UTR Description Root Leaf Leaf Leaf Petiole FlowerEXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.AC145767v28-1:1:2 9.52 3.52 5.37 3.972.12 EXP-BSAcVNV.FLT:1:2 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00 1.00EXP-BSAcVNV.FLT:1:2 T-Mt.AC145767v28-1:1:2 58.63 23.94 21.56 13.03 15.679.04 EXP-CERV.FLT:1:2 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00 1.00EXP-CERV.FLT:1:2 T-Mt.AC145767v28-1:1:2 17.75 4.70 6.79 16.86 17.7927.57 EXP-DaMV.FLT:1:2 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00 1.00EXP-DaMV.FLT:1:2 T-Mt.AC145767v28-1:1:2 11.94 8.52 7.70 7.23 9.51 4.55EXP-CUCme.eEF1a:1:1 T-Gb.FbL2-1:1:1 1.00 1.00 1.00 1.00 1.00 1.00EXP-CUCme.eEF1a:1:1 T-Mt.AC145767v28-1:1:2 12.15 2.70 5.77 7.23 12.473.11 EXP-Mt.Ubq2:1:2 T-Gb.FbL2-1:1:1 1.00 1.00 1.00 1.00 1.00 1.00EXP-Mt.Ubq2:1:2 T-Mt.AC145767v28-1:1:2 1.07 1.45 4.06 1.77 0.73 0.83

TABLE 27 Fold expression differences in R₁ generation transformedsoybean plants in Yellow Pod Embryo, Yellow Pod Cotyledon, R3 ImmatureSeed, R3 Pod and R5 Cotyledon. Yellow Yellow R3 Pod Pod Immature R3 R5EXP Description 3′ UTR Description Embryo Cotyledon Seed Pod CotyledonEXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00EXP-CaMV.35S-enh+Ph.DnaK:1:3 T-Mt.AC145767v28-1:1:2 7.48 24.12 14.675.33 5.59 EXP-BSAcVNV.FLT:1:2 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00EXP-BSAcVNV.FLT:1:2 T-Mt.AC145767v28-1:1:2 19.34 3.32 5.32 8.07 5.78EXP-CERV.FLT:1:2 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00EXP-CERV.FLT:1:2 T-Mt.AC145767v28-1:1:2 21.50 60.50 37.89 27.25 26.16EXP-DaMV.FLT:1:2 T-Gb.E6-3b:1:1 1.00 1.00 1.00 1.00 1.00EXP-DaMV.FLT:1:2 T-Mt.AC145767v28-1:1:2 26.84 15.76 1.53 4.33 6.26EXP-CUCme.eEF1a:1:1 T-Gb.FbL2-1:1:1 1.00 1.00 1.00 1.00 1.00EXP-CUCme.eEF1a:1:1 T-Mt.AC145767v28-1:1:2 5.14 6.47 3.60 8.56 4.56EXP-Mt.Ubq2:1:2 T-Gb.FbL2-1:1:1 1.00 1.00 1.00 EXP-Mt.Ubq2:1:2T-Mt.AC145767v28-1:1:2 0.62 1.51 4.86

As demonstrated in Tables 24 and 25, the Medicago 3′ UTRT-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1) boosted expression of the sixconstitutive EXP elements relative to T-Gb.E6-3b:1:1(SEQ ID NO: 40), butin different ways depending upon the specific EXP element and tissue.The EXP element, EXP-Mt.AC145767v28:1:1, when used to drive GUS andoperably linked to its native 3′ UTR T-Mt.AC145767v28-1:1:2 expressedvery low in all of the tissues assayed and was undetectable in R3Immature Seed, R3 Pod, and R1 Flower. Some tissues of plants comprisingthe EXP element EXP-Mt.Ubq2:1:2 and the 3′ UTR T-Mt.AC145767v28-1:1:2demonstrated reduced expression relative to the combination ofEXP-Mt.Ubq2:1:2 and T-Gb.FbL2-1:1:1. This reduced expression was seen inR3 Immature Seed, R1 Flower, and R1 Petiole while, in contrast, Vn5 SinkLeaf and R5 Cotyledon expression was enhanced greater than four-fold.There was no change in root expression (Vn5 Root) with EXP-Mt.Ubq2:1:2and either 3′ UTR.

The regulatory expression element groups EXP-CERV.FLT:1:2 andEXP-DaMV.FLT:1:2 provided the highest levels of expression. Asdemonstrated in Tables 26 and 27, these two EXPs were enhanced in alltissues with T-Mt.AC145767v28-1:1:2 relative to the same EXPs combinedwith T-Gb.E6-3b:1:1(SEQ ID NO: 40). The regulatory expression elementgroup EXP-CERV.FLT:1:2 was enhanced 60.50-fold in the developing YellowPod Cotyledon and less so in the Yellow Pod Embryo (21.50-fold), whilethe regulatory expression element group EXP-DaMV.FLT:1:2 was enhanced toa greater degree in the Yellow Pod Embryo than in the Yellow PodCotyledon (26.80- vs. 15.76-fold enhancement, respectively). Theseexpression and enhancement differences offer an opportunity to providetailored expression of a transgene in the later stage developing seed.The regulatory expression element group EXP-BSAcVNV.FLT:1:2 expressedhighest in the R3 Pod and Vn5 Root when combined with T-Gb.E6-3b:1:1(see Tables 25 and 26). The expression of EXP-BSAcVNV.FLT:1:2 in thesetwo tissues was enhanced dramatically when combined withT-Mt.AC145767v28-1:1:2, particularly in Vn5 Root. Further, theexpression of EXP-BSAcVNV.FLT:1:2 was boosted 58.63-fold when combinedwith T-Mt.AC145767v28-1:1:2 relative to this same EXP combined withT-Gb.E6-3b:1:19 (SEQ ID NO: 40)

In sum, the Medicago truncatula 3′ UTR T-Mt.AC145767v28-1:1:2 (SEQ IDNO: 1) enhanced expression of six different constitutive EXP elementswhich were derived from both plant and plant viral genomic DNA. Inaddition, this 3′ UTR enhanced expression of the seed-preferred EXPelement EXP-Gm.Sphas1:1:1 (SEQ ID NO: 54) relative to most of the otherMedicago-derived UTRs. Accordingly, this 3′ UTR is suited for providingenhanced expression of a promoter or combination of operably linkedexpression elements in a construct.

Example 7 Analysis of EXP-Mt.Ubq2:1:2 (SEQ ID NO: 31) in StablyTransformed Soybean Plants

Soybean plants were transformed with vectors, specifically plasmidconstructs, comprising the constitutive regulatory expression elementgroup EXP-Mt.Ubq2:1:2 (SEQ ID NO: 31) operably linked to a GUS codingsequence. These transformed plants were then assayed for GUS expressionin stably transformed soybean plants.

The plant vectors utilized in these experiments were built using cloningmethods known in the art. The resulting vectors comprised a left borderregion from A. tumefaciens; a first transgene expression cassette forselection of transformed plant cells that confers resistance to theantibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); asecond transgene expression cassette used to assess the activity ofEXP-Mt.Ubq2:1:2 (SEQ ID NO: 31) which comprised EXP-Mt.Ubq2:1:2 operablylinked 5′ to a coding sequence for ß-glucuronidase (GUS) that possessesa processable intron (GUS-2, SEQ ID NO: 44) operably linked 5′ to the 3′UTR T-Mt.AC145767v28-1:1:2 (SEQ ID NO: 1) derived from Medicagotruncatula, or the 3′ UTRs T-Gb.E6-3b:1:1 (SEQ ID NO: 40) orT-Gb.FbL2-1:1:1 (SEQ ID NO: 41) derived from Gossypium barbadense; and aright border region from A. tumefaciens.

The resulting vectors were used to transform soybean plants as describedin Example 3. Tables 28 and 29 show the average quantitative GUSexpression values assayed in various tissues and developmental timepoints for the stably transformed soybean plants.

TABLE 28 Average GUS expression in leaf, root and flower for stablytransformed soybean plants comprising EXP-Mt.Ubq2:1:2 (SEQ ID NO: 31).Vn5 Vn5 R1 Plasmid Vn5 Sink Source Source R1 R1 Construct 3′ UTRDescription Root Leaf Leaf Leaf Petiole Flower pMON125840 T-Gb.E6-3b:1:1252.58 126.69 86.01 49.05 108.41 83.23 pMON125841 T-Gb.FbL2-1:1:1 800.93202.73 275.48 143.6 1195.97 482.13 pMON155502 T-Mt.AC145767v28-1:1:2 855293.68 1118.76 254.25 875.67 398.1

TABLE 29 Average GUS expression in pod and seed tissues for stablytransformed soybean plants comprising EXP-Mt.Ubq2:1:2 (SEQ ID NO: 31).R3 Yellow Yellow Plasmid Immature R3 R5 Pod Pod Construct 3′ UTRDescription Seed Pod Cotyledon Embryo Cotyledon pMON125840T-Gb.E6-3b:1:1 2.22 111.19 3.21 24.31 50.98 pMON125841 T-Gb.FbL2-1:1:1400.15 875.75 72.66 129.84 83.45 pMON155502 T-Mt.AC145767v28-1:1:2247.18 1324.98 352.81

As demonstrated in Tables 28 and 29, EXP-Mt.Ubq2:1:2 (SEQ ID NO: 31) isable to drive constitutive expression of a transcribable DNA molecule instably transformed soybean plants. Further, different 3′ UTRs affect thedegree of expression in each tissue. For example, combiningEXP-Mt.Ubq2:1:2 with T-Gb.E6-3b:1:1 resulted in lower expression in allof the tissues assayed than the other two 3′ UTRs, T-Gb.FbL2-1:1:1 andT-Mt.AC145767v28-1:1:2. However, regardless of which 3′ UTR was applied,EXP-Mt.Ubq2:1:2 provides medium-to-high constitutive expression, thedegree of which can be modulated by a selection of which 3′ UTR isoperably linked to the EXP.

Example 8 Enhancers Derived from the Regulatory Elements

Enhancers may be derived from the promoter elements provided herein,such as SEQ ID NOs: 32 and 36. An enhancer element may be comprised ofone or more cis-regulatory elements that, when operably linked 5′ or 3′to a promoter element, or operably linked 5′ or 3′ to additionalenhancer elements that are operably linked to a promoter, can enhance ormodulate expression of a transcribable DNA molecule, or provideexpression of a transcribable DNA molecule in a specific cell type orplant organ or at a particular time point in development or circadianrhythm. Enhancers are made by removing the TATA box or functionallysimilar elements and any downstream sequence that allow transcription tobe initiated from the promoters or promoter fragments.

Enhancer elements may be derived from the promoter elements providedherein and cloned using methods known in the art to be operably linked5′ or 3′ to a promoter element, or operably linked 5′ or 3′ toadditional enhancer elements that are operably linked to a promoter.Alternatively, enhancer elements maybe cloned, using methods known inthe art, to be operably linked to one or more copies of the enhancerelement which are operably linked 5′ or 3′ to a promoter element, oroperably linked 5′ or 3′ to additional enhancer elements that areoperably linked to a promoter. Further, enhancer elements can be clonedto be operably linked 5′ or 3′ to a promoter element derived from adifferent genus organism, or operably linked 5′ or 3′ to additionalenhancer elements derived from other genus organisms or the same genusorganism that are operably linked to a promoter derived from either thesame or different genus organism, resulting in a chimeric regulatoryelement. A GUS expression plant transformation vector maybe constructedusing methods known in the art similar to the constructs described inthe previous Examples in which the resulting plant expression vectorscontain a left border region from A. tumefaciens; a first transgeneselection cassette that confers resistance to an antibiotic or herbicideand is utilized for selection of transformed plant cells; and a secondtransgene cassette in which an enhancer element is operably linked to apromoter forming a chimeric promoter element, which is operably linked5′ to a leader element, which is operably linked 5′ to a coding sequencefor GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44),operably linked to a 3′ UTR such as T-Gb.E6-3b:1:1 or any of thosedescribed above from Medicago truncatula; and a right border region fromA. tumefaciens.

GUS expression driven by a regulatory element comprising one or moreenhancers maybe evaluated in stable or transient plant assays asdescribed herein to determine the effects of the enhancer element onexpression of a transcribable DNA molecule. Modifications to one or moreenhancer elements or duplication of one or more enhancer elements maybeperformed based upon empirical experimentation, and the resulting geneexpression regulation that is observed using each regulatory elementcomposition. Altering the relative positions of one or more enhancers inthe resulting regulatory or chimeric regulatory elements may affect thetranscriptional activity or specificity of the regulatory or chimericregulatory element and is determined empirically to identify the bestenhancers for the desired transgene expression profile within a plant.

Example 9 Analysis of the Effect of 3′ UTRs on Constitutive GUSExpression in Stably Transformed Corn Plants

Corn plants were transformed with binary plasmid constructs to assessthe effect of the Medicago 3′ UTR T-Mt.Oxr-1:2:1 (SEQ ID NO: 17) onexpression relative to two 3′ UTRs used frequently in corn plants.Specifically, the corn plants were transformed with vectors containingan EXP that exhibited a constitutive expression profile drivingexpression of the ß-glucuronidase (GUS) transgene, which was operablylinked to the Medicago 3′ UTR T-Mt.Oxr-1:2:1 (SEQ ID NO: 17). Thesetransformed corn plants were compared to transformed corn plants inwhich GUS was operably linked to either the 3′ UTR T-AGRtu.nos-1:1:13(SEQ ID NO: 49) or the 3′ UTR T-Os.LTP:1 (SEQ ID NO: 56).

The binary plasmid constructs utilized in these experiments were builtusing cloning methods known in the art. The resulting vectors containeda right border region from A. tumefaciens; a first expression cassetteto assay the 3′ UTR sequence wherein a constitutive regulatoryexpression element group EXP-FMV.35S-enh+Ta.Lhcb1+Zm.DnaK:1:2 (SEQ IDNO: 56) is operably linked 5′ to a coding sequence for GUS thatpossesses a processable intron (GUS-2, SEQ ID NO: 44), which is operablylinked 5′ to one of the following three 3′ UTRs: T-Mt.Oxr-1:2:1 (SEQ IDNO: 17), T-AGRtu.nos-1:1:13 (SEQ ID NO: 49) or T-Os.LTP:1 (SEQ ID NO:56); a second transgene expression cassette used for selection oftransformed plant cells that confers resistance to the herbicideglyphosate (driven by the rice Actin 1 promoter); and a left borderregion from A. tumefaciens. The resulting plasmids were used totransform corn plants.

Histochemical GUS analysis was used for qualitative expression analysisof the transformed plants. Whole tissue sections were incubated with GUSstaining solution X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) (1mg/ml) for an appropriate length of time, rinsed, and visually inspectedfor blue coloration. GUS activity was qualitatively determined by directvisual inspection or inspection under a microscope using selected plantorgans and tissues. The R₀ plants were inspected for expression in theroots and leaves, as well as the anther, silk, and developing seed andembryo, 21 days after pollination (21 DAP).

Quantitative analysis for the transformed corn plants was alsoperformed. For the quantitative analysis, total protein was extractedfrom selected tissues of the transformed corn plants. One microgram oftotal protein was used with the fluorogenic substrate4-methyleumbelliferyl-β-D-glucuronide (MUG) in a total reaction volumeof 50 μl. The reaction product, 4-methlyumbelliferone (4-MU), ismaximally fluorescent at high pH, where the hydroxyl group is ionized.Addition of a basic solution of sodium carbonate simultaneously stopsthe assay and adjusts the pH for quantifying the fluorescent product.Fluorescence is measured with excitation at 365 nm, emission at 445 nmusing a Fluoromax-3 (Horiba; Kyoto, Japan) with Micromax Reader, withslit width set at excitation 2 nm and emission 3 nm.

Table 30 shows the average quantitative GUS expression measureddemonstrating different effects of each 3′ UTR on the same constitutiveexpressing EXP.

TABLE 30 Average GUS expression in corn plants transformed withdifferent 3′ UTRs. Plasmid Construct pMON128881 pMON119693 pMON132035Developmental T-Mt.Oxr-1:2:1 T-Os.LTP:1 T-AGRtu.nos:13 Stage Tissue (SEQID NO: 17) (SEQ ID NO: 56) (SEQ ID NO: 49) V4 Leaf 205 232 222 Root 126134 44 V7 Leaf 277 534 293 Root nd 135 nd VT Leaf 314 429 194 Root 1981043 291 Flower/Anther 527 486 308 R1 Cob/Silk 169 1258 319 R3 Embryo21DAP 179 72 101 Endosperm 21DAP 516 207 243

As can be seen in Table 30, each 3′ UTR had a different effect onconstitutive expression driven by EXP-FMV.35S-enh+Ta.Lhcb1+Zm.DnaK:1:2(SEQ ID NO: 56). For example, the 3′ UTR T-Os.LTP:1 (SEQ ID NO: 56)appeared to enhance expression in the VT Root and R1 Cob/Silk relativeto the other two 3′ UTRs. The 3′ UTR T-Mt.Oxr-1:2:1 (SEQ ID NO: 17)appeared to enhance expression in the R3 seed, both in the 21DAPendosperm and 21DAP Embryo relative to T-AGRtu.nos-1:1:13 (SEQ ID NO:49) and T-Os.LTP:1 (SEQ ID NO: 56). Expression in the Flower/Anther wasalso higher using T-Mt.Oxr-1:2:1 (SEQ ID NO: 17) relative to the othertwo 3′ UTRs. The differences in expression observed for each of the 3′UTRs demonstrates the usefulness of each 3′ UTR in modulatingexpression. Thus, these experiments demonstrate that the selection of a3′ UTR can be used in transgene cassettes to fine tune expression of aparticular transcribable DNA molecule. This experiment also demonstratesthe ability of a dicot-derived 3′ UTR, such as T-Mt.Oxr-1:2:1, to affecttranscription in a monocot species such as corn.

Example 10 Analysis of Intron Enhancement of GUS Activity Using PlantDerived Protoplasts

Generally, an intron is selected based upon experimentation andcomparison with an intronless vector control to empirically select anintron and configuration within the vector transfer DNA (T-DNA) elementarrangement for optimal expression of a transgene. For example, in theexpression of an herbicide resistance gene, such as CP4 (U.S. RE39247),which confers tolerance to glyphosate, it is desirable to have transgeneexpression within the reproductive tissues as well as the vegetativetissues in order to prevent the loss of yield when applying theherbicide. An intron in this instance would be selected upon itsability, when operably linked to a constitutive promoter, to enhanceexpression of the herbicide resistance conferring transgene,particularly within the reproductive cells and tissues of the transgenicplant, and thus providing both vegetative and reproductive tolerance tothe transgenic plant when sprayed with the herbicide. In most ubiquitingenes, the 5′ UTR is comprised of a leader, which has an intron sequenceembedded within it. The regulatory elements derived from such genes aretherefore assayed using the entire 5′ UTR comprising the promoter,leader, and intron. To achieve different expression profiles or tomodulate the level of transgene expression, the intron from such aregulatory element may be removed or substituted with a heterologousintron.

The intron presented herein as SEQ ID NO: 34 was identified usinggenomic DNA contigs in comparison to expressed sequence tag clusters, orcDNA contigs, to identify exon and intron sequences within the genomicDNA. In addition, 5′ UTR or leader sequences were also used to definethe intron/exon splice junction of one or more introns under conditionswhen the gene sequence encodes a leader sequence that is interrupted byone or more introns. Introns were cloned using methods known in the artinto a plant transformation vector to be operably linked 3′ to aregulatory element and leader fragment and operably linked 5′ to eithera second leader fragment or to coding sequences, such as the expressioncassettes presented in FIG. 1.

Thus, for example, a first possible expression cassette, such asExpression Cassette Configuration 1 in FIG. 1, is comprised of apromoter or chimeric promoter element [A], operably linked 5′ to aleader element [B], operably linked 5′ to a test intron element [C],operably linked to a coding region [D], which is operably linked to a 3′UTR element [E]. Alternatively, a second possible expression cassette,such as Expression Cassette Configuration 2 in FIG. 1, is comprised of apromoter or chimeric promoter element [F], operably linked 5′ to a firstleader element or first leader element fragment [G], operably linked 5′to a test intron element [H], operably linked 5′ to a second leaderelement or first leader element second fragment [I], operably linked toa coding region [J], which is operably linked to a 3′ UTR element [K].Further, a third possible expression cassette, such as ExpressionCassette Configuration 3 in FIG. 1, is comprised of a promoter orchimeric promoter element [L], operably linked 5′ to a leader element[M], operably linked 5′ to a first fragment of the coding sequenceelement [N], operably linked 5′ to an intron element [O] element,operably linked 5′ to a second fragment of the coding sequence element[P], which is operably linked to a 3′ UTR element [Q]. Notably,Expression Cassette Configuration 3 is designed to allow splicing of theintron in such a manner as to produce a complete open reading framewithout a frame shift between the first and second fragment of thecoding sequence.

As discussed herein, it may be preferable to avoid using the nucleotidesequence AT or the nucleotide A just prior to the 5′ end of the splicesite (GT) and the nucleotide G or the nucleotide sequence TG,respectively just after 3′ end of the splice site (AG) to eliminate thepotential of unwanted start codons from being formed during processingof the messenger RNA into the final transcript. The DNA sequence aroundthe 5′ or 3′ end splice junction sites of the intron can thus bemodified.

Introns may be assayed for an enhancement effect through the ability toenhance expression in transient assay or stable plant assay. Fortransient assay of intron enhancement, a base plant vector isconstructed using methods known in the art. The intron is cloned into abase plant vector which comprises an expression cassette comprised of aconstitutive EXP comprised of a promoter and leader such asEXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO: 42), operably linked 5′ to atest intron element (e.g. one SEQ ID NO: 34), operably linked to acoding sequence for GUS that possesses a processable intron (GUS-2, SEQID NO: 44), operably linked to the 3′ UTR from (T-Gb.E6-3b:1:1, SEQ IDNO: 40). Protoplast cells derived from soybean or other genus planttissue can be transformed with the base plant vector and Luciferasecontrol vectors as described previously in Example 2 above, and assayedfor activity. To compare the relative ability of the intron to enhanceexpression, GUS values are expressed as a ratio of GUS to Luciferaseactivity and compared with those levels imparted by a constructcomprising the constitutive promoter operably linked to a known intronstandard such as that as the intron derived from the Nicotiana tabacumelongation factor 4A10 gene, I-Nt.eIF4A10-1:1:1 (SEQ ID NO: 57), as wellas a construct comprising the constitutive promoter, but without anintron operably linked to the promoter.

For stable plant assay of the intron presented as SEQ ID NO: 34, a GUSexpression plant transformation vector can be constructed similar to theconstructs described in the previous examples in which the resultingplant expression vectors contains a right border region from A.tumefaciens; a first expression cassette comprised of a constitutive EXPcomprised of a promoter and leader such as EXP-CaMV.35S-enh+Ph.DnaK:1:3(SEQ ID NO: 42), operably linked 5′ to a test intron element (e.g., SEQID NO: 34), operably linked to a coding sequence for GUS that possessesa processable intron (GUS-2, SEQ ID NO: 44), operably linked to the 3′UTR from Gossypium barbadense (T-Gb.E6-3b:1:1, SEQ ID NO: 40).Protoplast cells derived from corn or other genus plant tissue may betransformed with the base plant vector and luciferase control vectors,as described previously in Example 2 above, and assayed for activity. Tocompare the relative ability of the intron to enhance expression, GUSvalues are expressed as a ratio of GUS to luciferase activity andcompared with those levels imparted by a construct comprising theconstitutive promoter operably linked to a known intron standard such asthat as the intron derived from the Nicotiana tabacum elongation factor4A10 gene, I-Nt.eIF4A10-1:1:1 (SEQ ID NO: 57), as well as a constructcomprising the constitutive promoter, but without an intron operablylinked to the promoter.

It should be noted that the intron presented as SEQ ID NO: 34 can bemodified in a number of ways, such as deleting fragments within theintron sequence, which may reduce expression or duplication of fragmentswith the intron that may enhance expression. In addition, DNA sequenceswithin the intron that may affect the specificity of expression toeither particular cells types or tissues and organs can be duplicated oraltered or deleted to affect expression and patterns of expression ofthe transgene. In addition, the intron provided herein can be modifiedto remove any potential start codons (ATG) that may cause unintentionaltranscripts from being expressed from improperly spliced introns asdifferent, longer or truncated proteins. Once the intron has beenempirically tested, or it has been altered based upon experimentation,the intron may be used to enhance expression of a transgene in stablytransformed plants that can be of any genus monocot or dicot plant, solong as the intron provides enhancement of the transgene. The intron canalso be used to enhance expression in other organisms, such as algae,fungi, or animal cells, so long as the intron provides enhancement orattenuation or specificity of expression of the transgene to which it isoperably linked.

Having illustrated and described the principles of the invention, itshould be apparent to persons skilled in the art that the invention canbe modified in arrangement and detail without departing from suchprinciples. We claim all modifications that are within the spirit andscope of the claims. All publications and published patent documentscited herein are hereby incorporated by reference to the same extent asif each individual publication or patent application is specifically andindividually indicated to be incorporated by reference.

1. A recombinant DNA molecule comprising a DNA sequence selected fromthe group consisting of: a) a DNA sequence with at least 85 percentsequence identity to SEQ ID NO:30; b) a DNA sequence comprising SEQ IDNO:30; and c) a fragment of SEQ ID NO:30, wherein the fragment hasgene-regulatory activity; wherein said DNA sequence is operably linkedto a heterologous transcribable DNA molecule.
 2. The recombinant DNAmolecule of claim 1, wherein said DNA sequence has at least 90 percentsequence identity to the DNA sequence of SEQ ID NO:30.
 3. Therecombinant DNA molecule of claim 1, wherein said DNA sequence has atleast 95 percent sequence identity to the DNA sequence of SEQ ID NO:30.4. The recombinant DNA molecule of claim 1, wherein the heterologoustranscribable polynucleotide molecule comprises a gene of agronomicinterest.
 5. The recombinant DNA molecule of claim 4, wherein the geneof agronomic interest confers herbicide tolerance in plants.
 6. Therecombinant DNA molecule of claim 4, wherein the gene of agronomicinterest confers pest resistance in plants.
 7. A transgenic plant cellcomprising a recombinant DNA molecule comprising a DNA sequence selectedfrom the group consisting of: a) a DNA sequence with at least 85 percentsequence identity to SEQ ID NO:30; b) a DNA sequence comprising SEQ IDNO:30; and c) a fragment of SEQ ID NO:30, wherein the fragment hasgene-regulatory activity; wherein said DNA sequence is operably linkedto a heterologous transcribable DNA molecule.
 8. The transgenic plantcell of claim 7, wherein said transgenic plant cell is amonocotyledonous plant cell.
 9. The transgenic plant cell of claim 7,wherein said transgenic plant cell is a dicotyledonous plant cell.
 10. Atransgenic plant, or part thereof, comprising a recombinant DNA moleculecomprising a DNA sequence selected from the group consisting of: a) aDNA sequence with at least 85 percent sequence identity to SEQ ID NO:30;b) a DNA sequence comprising SEQ ID NO:30; and c) a fragment of SEQ IDNO:30, wherein the fragment has gene-regulatory activity; wherein saidDNA sequence is operably linked to a heterologous transcribable DNAmolecule.
 11. A progeny plant of the transgenic plant of claim 10, or apart thereof, wherein the progeny plant or part thereof comprises saidrecombinant DNA molecule.
 12. A transgenic seed of the transgenic plantof claim 10 wherein the transgenic seed comprises the recombinant DNAmolecule.
 13. A method of producing a commodity product comprisingobtaining a transgenic plant or part thereof according to claim 10 andproducing the commodity product therefrom.
 14. The method of claim 13,wherein the commodity product is protein concentrate, protein isolate,grain, starch, seeds, meal, flour, biomass, or seed oil.
 15. A method ofproducing a transgenic plant comprising: a) transforming a plant cellwith the recombinant DNA molecule of claim 1 to produce a transformedplant cell; and b) regenerating a transgenic plant from the transformedplant cell.